JP5857312B2 - Diagnostic marker for heart disease - Google Patents

Description

  The present invention relates to a method for evaluating the degree of oxidation of a marker protein, which is contained as a soluble component in blood, and reflects the degree of progression of heart disease, particularly ischemic heart disease, and oxidation of the marker protein The present invention relates to an enzyme-digested peptide fragment derived from the marker protein, which is used to evaluate the degree of progression of the marker protein. More specifically, the present invention relates to oxidized low density lipoprotein (LDL; low), which is a marker protein contained as a soluble component in blood, reflecting the progression of ischemic heart disease. -density lipoprotein), in particular, a method for evaluating the degree of oxidation of Apolipoprotein B100 (ApoB100 protein) contained in malondialdehyde LDL (MDA-LDL), and oxidation of ApoB100 protein contained in MDA-LDL The present invention relates to an enzyme-digested peptide fragment derived from ApoB100 protein that has undergone oxidative denaturation, and is used for evaluation of the degree of progression of the protein.

  Due to recent rapid progress in molecular biological technology, information on biomolecules involved in diseases, which has not been obtained in the past, is rapidly accumulating. Among them, detailed information on biomolecules contained in clinical samples collected from patients suffering from diseases reflects the pathological aspects of individual diseases. Therefore, it is expected that detailed information on biomolecules that reflect the pathological aspects of individual diseases can be applied to the prevention, diagnosis, development of treatment methods, and prediction of sensitivity to treatment. Specifically, research for personalized medicine using information on biomolecules involved in target diseases has been actively conducted. In recent years, lifestyle-related diseases have been highlighted, and the importance of identifying biomolecules involved in lifestyle-related diseases and understanding the pathological conditions at the molecular level based on information on the identified biomolecules has been recognized.

  Arteriosclerotic disease is one of lifestyle-related diseases. In fact, arteriosclerotic diseases are currently the leading cause of death in developed countries (2nd in Japan, 15.9% heart disease, 11.8% cerebrovascular disease, 1st in the US) Preventive measures are an important medical and social issue. In the process of progression and deterioration of atherosclerosis, particularly atherosclerosis, LDL loses affinity for LDL receptor as it is oxidized, denatured and glycated, while macrophages The process of binding LDL that has undergone oxidation, denaturation, and glycation via a scavenger receptor, taking it into cells, and forming foam cells plays a central role. Therefore, in LDL contained as a soluble component in blood, the concentration of LDL that has undergone oxidation, denaturation, and saccharification, in particular, the concentration of oxidized LDL, and the development of a technique for detecting the oxidation site, It is considered possible to understand the pathological condition at the molecular level in atherosclerosis.

  In fact, from the study of the relationship between arteriosclerosis and oxidized LDL, oxidative stress, which increases the concentration of oxidized LDL in plasma, is central in the pathogenesis of atherosclerosis and cardiovascular diseases caused by arteriosclerosis. It has been suggested to play a role (see Non-Patent Document 1 and Non-Patent Document 2). Therefore, for the purpose of grasping the pathological conditions of arteriosclerosis and cardiovascular disease caused by arteriosclerosis, high-precision measurement of oxidized LDL concentration in clinical samples is significant.

  An ELISA (Enzyme-Linked Immunosorbent Assay) method using an antibody specific for oxidized LDL and modified LDL has been reported as a method for selectively measuring the content of oxidized LDL and modified LDL coexisting with LDL. (See Non-Patent Document 1 and Patent Document 1).

The ELISA method is a technique widely used for detecting and quantifying the concentration of an antibody or antigen contained in a sample. In particular, it is suitable for the purpose of selectively detecting and quantifying an antigen contained in a sample containing a large number of contaminants by using an antibody specific to the target antigen. For this reason, it is widely used in screening tests for the purpose of quantifying the presence or absence of proteins related to the target disease contained in clinical samples. For example, in the sandwich-ELISA method, using the two types of antibodies specific to the target antigen (target protein), the target antigen (target protein) can be obtained by the following procedures (1) to (6). Detection is taking place.
(1) An antibody (capture antibody) against a target protein (antigen) is adsorbed on a solid phase.
(2) Block the solid phase with skim milk or the like.
(3) A primary antibody that recognizes an epitope different from the sample solution and the capture antibody is added to the solid phase. At this point, a solid phase-capture antibody-antigen-primary antibody complex is formed on the solid surface.
(4) Wash away unreacted antigen and primary antibody.
(5) When the enzyme is not labeled on the primary antibody, the enzyme-labeled secondary antibody is allowed to act. Thereafter, excess secondary antibody is washed away.
(6) A label enzyme substrate (usually a color developing or luminescent reagent) is added to detect the product of the enzyme reaction.

  Since the sandwich-ELISA method uses a labeling enzyme to detect the product of the enzyme reaction of the labeling enzyme, highly sensitive detection is possible, so the antigen (target protein) contained in a trace amount in the sample can be detected. Widely adopted for quantitative detection. However, the site (epitope) recognized by the antibody is a specific part of the antigen (target protein). Therefore, when oxidation or denaturation occurs at a site other than the epitope region of the antigen (target protein) and the state of the antigen (target protein) changes over time, it is difficult to trace the change over time. There are many.

Japanese Patent No. 315587

ITABE Hiroykuki, (2002), YAKIGAKU ZASSHI, Vol.122, No.10, p.745-753 Fogelman, AM, Shechter, I., Seager, J., Hokom, M., Childe, JS, Edward, PA, (1980 April) Proc. Natl. Acad. Sci. USA, Vol. 77, No. 4, p. .2214-2218

  The method for detecting and quantifying the target antigen (target protein) using the sandwich-ELISA method described above is that the capture antibody recognition site (first epitope) and primary antibody recognition site (second epitope) are specific. Therefore, it cannot be determined whether or not oxidation or denaturation has occurred in a region other than these two recognition sites.

  Therefore, among oxidized LDL and denatured LDL, it is used for the purpose of quantifying the total amount of oxidized LDL and denatured LDL that retain both the capture antibody recognition site (first epitope) and the primary antibody recognition site (second epitope). ing. Specifically, a specific antibody that recognizes oxidized LDL and denatured LDL but does not recognize LDL as a capture antibody, while an antibody that recognizes any of oxidized LDL, denatured LDL, and LDL as a primary antibody. Has been reported (see Non-Patent Document 1 and Patent Document 1).

As a specific antibody that recognizes the oxidized LDL and the modified LDL but does not recognize LDL, for example, an antioxidant LDL monoclonal antibody “DLH3” has been reported (see Non-Patent Document 1). The “DLH3” antibody does not bind to untreated LDL, acetylated LDL, MDA-LDL, or glycated LDL, and is also a phospholipid phosphatidylcholine (PC) contained in LDL, or other lipid (cholesterol ester). Etc.), and aldehyde-type oxidized PC generated by oxidation of PC. Furthermore, as a specific antibody that recognizes the oxidized LDL and modified LDL but does not recognize LDL, for example, a monoclonal antibody against human MDA-LDL has also been reported (see Patent Document 1). Human MDA-LDL is obtained by binding malondialdehyde (MDA) to ApoB100 protein, which is a protein serving as the nucleus of LDL. In the oxidation process of LDL, for example, various aldehyde molecules including MDA are generated as an oxidation product of the carbon chain of the highly unsaturated fatty acid along with the peroxidation process of PC. The produced MDA can undergo keto-enol tautomerism of CH ₂ (CHO) ₂ ⇔HO—CH═CH—CHO, and the amino group (—NH ₂₎ on the side chain of the Lys residue contained in the protein. ), For example, modification of the shape of —NH—CH═CH—CHO is performed.

  Therefore, in oxidized LDL, at least oxidation of contained phospholipids such as PC and MDA conversion to ApoB100 protein proceed. The binding between LDL and the LDL receptor is due to the binding of ApoB100 protein to the LDL receptor. When the MDA conversion to the ApoB100 protein proceeds, the ability of the ApoB100 protein to bind to the LDL receptor is impaired. On the other hand, macrophages bind to oxidized LDL through their scavenger receptors and take up into cells to form foam cells.

  As preventive measures against lifestyle-related diseases are listed as one of the policies, the effects of such measures will greatly affect both socially and economically. Among them, arteriosclerosis is a major cause of ischemic heart disease, cerebral infarction and the like. Therefore, preventive measures against arteriosclerosis are important medical and social issues.

  Arteriosclerosis is thought to proceed by the following mechanism. Cholesterol esters accumulate in the vascular endothelium. Smooth muscle cells, connective tissue, and the like also gather at the site where accumulation of cholesterol ester occurs. Next, a state called an atheroma is formed, and the thickness further increases with the formation of the atheroma. Eventually, at the site of atheroma formation, the blood vessels harden and the function of the arteries decreases.

  During the progression of arteriosclerosis, foam cells are mainly involved in the stage of atheroma formation. Foam cells involved in the formation of atheroma have been reported to be generated by macrophages taking up oxidized LDL. Uptake of oxidized LDL by macrophages is due to a mechanism in which oxidized LDL that has lost its affinity for LDL receptor is bound to the oxidized LDL via the scavenger receptor and taken into the cell. That is, the oxidized LDL has a shape of MDA-LDL formed by binding MDA to ApoB100, which is a main component protein of LDL. Therefore, the result which verifies that oxidized LDL, especially MDA-LDL is concerned in the early lesion of arteriosclerosis has been reported (refer nonpatent literature 2).

  Considering the above mechanism of progression of arteriosclerosis, an ELISA method using an anti-MDA-LDL antibody has been put into practical use for the purpose of detecting and quantifying oxidized LDL in blood, particularly MDA-LDL. (See Patent Document 1). However, detection of oxidized LDL by ELISA using an anti-MDA-LDL antibody is mainly aimed at grasping the blood concentration of oxidized LDL, and in particular, oxidized sites in oxidized LDL, particularly MDA-LDL and ApoB100 protein. It cannot be used to specify (MDA site). Therefore, in oxidized LDL, particularly in MDA-LDL, the oxidized site (MDA site) in ApoB100 protein, arteriosclerosis, and pathological conditions of heart diseases caused by atherosclerosis including ischemic heart disease It cannot be used for association with. The reason is that the capture antibody used in the examination by ELISA recognizes a specific site (epitope) of the antigen (target protein) with high selectivity, and overlooks a portion other than the specific site (epitope). This is because it is originally unsuitable.

  From the above situation, at present, there are few clear reports on the relationship between the modified state of the ApoB100 protein in oxidized LDL and so-called clinical information such as details of the degree of progression of arteriosclerosis and the onset mechanism of arteriosclerosis.

  As a result of considering the present situation, the present inventors have found that the following problems exist.

  The progression of arteriosclerosis, in particular, the progression of atherosclerosis is the result of oxidation of LDL by oxidative stress, an increase in the blood concentration of oxidized LDL, and the modification (MDA conversion) to ApoB100 protein in oxidized LDL, This is due to the loss of affinity for the LDL receptor. Therefore, in addition to measuring the blood concentration of oxidized LDL, measuring the modification (MDA) state in ApoB100 protein in oxidized LDL makes it possible to estimate the level of oxidative stress. In addition, measurement of blood concentration of oxidized LDL and measurement of modification (MDA) state of ApoB100 protein in oxidized LDL are performed simultaneously, so-called clinical information such as the progress of arteriosclerosis and details of the onset mechanism of arteriosclerosis. When it is found that the relation is found, it is useful for the development of clinical examination means effective in rationally estimating the degree of progression of arteriosclerosis.

  The present invention solves the aforementioned problems. That is, the object of the present invention is to clearly change the probability of oxidation and modification reflecting the degree of progression of arteriosclerosis among the oxidation sites and modification sites (MDA sites) in ApoB100 protein in oxidized LDL. It is to specify a group of specific “oxidation sites, modification sites (MDA sites)”. At the same time, the object of the present invention is to identify a specific group of “oxidation sites and modification sites (MDA sites)” and to oxidize and modify each “oxidation site and modification site (MDA site)”. To provide a method for detecting the oxidation or modification state of the specific “oxidation site, modification site (MDA site)”, which can easily and quantitatively detect whether or not there is a specific “oxidation site, modification site (MDA site)” It is in.

  In addition, the object of the present invention is to utilize the specific “oxidation site, modification site (MDA site)” oxidation and modification state detection method for the ApoB100 protein in oxidized LDL, and to detect the specific “ An object of the present invention is to provide a method for measuring the ratio of oxidation sites and modification sites (MDA sites) to oxidation and modification, and statistically estimating the progress of arteriosclerosis based on the measured ratios.

  Conventionally, arteriosclerosis indicating atherosclerosis, and further, heart disease caused by atherosclerosis, especially the degree of progression of ischemic heart disease, that is, the number of coronary lesions (ACI-TIPI score) Often, when performing assessments, catheterization has been used to assess the extent of atherosclerosis progression in the coronary arteries.

  Therefore, an object of the present invention is included in the blood sample using a blood sample collected from a patient instead of a catheter test used for evaluating the degree of progression of atherosclerosis in the coronary artery. In the ApoB100 protein in oxidized LDL, the ratio of the specific “oxidation site, modification site (MDA site)” undergoing oxidation and modification is measured, and the number of coronary lesions is evaluated based on the measured ratio It is an object of the present invention to provide a method for statistically estimating the progress of arteriosclerosis.

  In order to solve the above problems, the present inventors have a probability of undergoing oxidation and modification reflecting the degree of progression of arteriosclerosis among oxidation sites and modification sites (MDA sites) in ApoB100 protein in oxidized LDL. A group of specific “oxidation sites, modification sites (MDA sites)” that clearly change was identified.

  First, the present inventors made the following predictions.

  The increase in blood concentration of oxidized LDL is caused by the oxidation of LDL due to oxidative stress. In the process, various aldehyde molecules including MDA are generated as oxidation products with the oxidation of the carbon chain of fatty acid, particularly the carbon chain of highly unsaturated fatty acid, constituting the lipid contained in LDL. Depending on the level of oxidative stress, the amount of aldehyde molecules produced, for example the amount of MDA, varies. MDA reacts with the amino group of the Lys residue side chain in the ApoB100 protein to convert to MDA, but when the amount of MDA produced increases, the number of Lys residues converted to MDA increases. That is, depending on the level of oxidative stress, not only the blood concentration of oxidized LDL varies, but also the total number of MDA-modified Lys residues (MDA-modified sites) in the ApoB100 protein in the generated oxidized LDL varies. Is expected to.

In addition, in the ApoB100 protein in the generated oxidized LDL, oxidation of the sulfur atom of the side chain —CH ₂ —S—CH ₃ of the Met residue also proceeds. As the level of oxidative stress increases, the number of Met residues that undergo oxidation increases. That is, depending on the level of oxidative stress, not only the blood concentration of oxidized LDL varies, but also the total number of oxidized Met residues (oxidized sites) in the ApoB100 protein in the generated oxidized LDL varies. It is also expected to do.

  At that time, the MDA-modified Lys residue found in the ApoB100 protein in the oxidized LDL produced under the condition where the level of oxidative stress is low is the ApoB100 protein in the oxidized LDL produced under the condition where the level of oxidative stress is high. Similarly, it is an MDA-modified Lys residue. On the other hand, a plurality of MDA-modified Lys residues that are not found in the ApoB100 protein in the oxidized LDL produced under the condition where the level of oxidative stress is low are oxidized LDL produced under the condition where the level of oxidative stress is high. It is expected to be present in the middle ApoB100 protein.

  Similarly, the oxidized Met residue (oxidation site) found in the ApoB100 protein in oxidized LDL produced under conditions with low levels of oxidative stress is oxidized LDL produced under conditions with high levels of oxidative stress. Similarly, in the ApoB100 protein in the middle, it is an oxidized Met residue. On the other hand, a plurality of oxidized Met residues, which are not found in the ApoB100 protein in the oxidized LDL produced under the situation where the level of oxidative stress is low, are present in the oxidized LDL produced under the situation where the level of oxidative stress is high. Is expected to be present in the ApoB100 protein.

  Next, attention was paid to the fact that a reported monoclonal antibody against human MDA-LDL (anti-MDA-LDL monoclonal antibody) selectively binds to MDA-modified ApoB100 protein in oxidized LDL. Specifically, among MDA-modified Lys residues present in the MDA-modified ApoB100 protein in oxidized LDL, a specific MDA-modified Lys residue is included in the epitope of the anti-MDA-LDL monoclonal antibody. Focused on being. At that time, the anti-MDA-LDL monoclonal antibody contains MDA-modified ApoB100 protein in oxidized LDL that is produced under a condition where the level of oxidative stress is low, and in the oxidized LDL that is produced under a condition where the level of oxidative stress is high. When binding to both ApoB100 proteins, the specific MDA-modified Lys residue contained in the epitope does not depend on the level of oxidative stress, and is commonly subjected to MDA conversion (common MDA-ized site). It is judged that it is one of.

  Therefore, the anti-MDA-LDL monoclonal antibody used for the quantification of oxidized LDL contained in blood does not depend on the level of oxidative stress, and commonly has Lys residues (common MDA sites) that undergo MDA conversion. ) Is included in the epitope. In other words, when the immunoprecipitation method using the anti-MDA-LDL monoclonal antibody as a capture antibody is applied, Lys residue (common MDA site) that is commonly subjected to MDA conversion without depending on the level of oxidative stress. It is judged that oxidized LDL (MDA-LDL) containing MDA-modified ApoB100 protein having one of the above can be selectively separated.

On the other hand, conventionally, when measuring the blood concentration of oxidized LDL, plasma (blood plasma), or cellular components, fibrinogen, and coagulation factors are removed from the collected blood sample. A blood serum is prepared. Next, the concentration of oxidized LDL contained in the plasma sample or serum sample is measured by ELISA. The plasma sample or serum sample contains unoxidized LDL and oxidized LDL. Even in a situation where the level of oxidative stress is high, the ratio between the non-oxidized LDL content concentration C _LDL and the oxidized LDL content concentration C _OxLDL remains in the range of (C _OxLDL / C _LDL ) <1 ng / μg _LDL . In fact, AMI measured in patients with acute myocardial infarction (AMI) with a high number of coronary lesions (C _OxLDL / C _LDL ) and _AMI measured in healthy individuals with a low number of coronary lesions (C _OxLDL / C _LDL ) _{When normal} is compared, it is reported that the average value thereof is (C _OxLDL / C _LDL ) _AMI / (C _OxLDL / C _LDL ) _normal is about 3.5 (see Non-Patent Document 1).

  Oxidized LDL, which is separated by applying immunoprecipitation, is actually a mixture of oxidized LDL with a range of distributions in the degree of oxidation. Depending on the level of oxidative stress, the distribution of the degree of oxidation in the resulting mixture of oxidized LDL is different. Due to the difference in the distribution of the degree of oxidation, there is a difference in the distribution of the total number of MDA-modified Lys residues in the ApoB100 protein in the generated oxidized LDL and the distribution of the total number of oxidized Met residues. is expected.

  By applying an immunoprecipitation method using an antibody-immobilized bead in which an anti-MDA-LDL monoclonal antibody is immobilized on the surface of a bead carrier, for example, a magnetic bead, MDA-LDL was selectively bound to the antibody in oxidized LDL. Thereafter, when the beads are recovered by solid-liquid separation and washed, only MDA-LDL can be separated from the oxidized LDL.

  Oxidized LDL (MDA-LDL), which is selectively bound to anti-MDA-LDL monoclonal antibody, is composed of MDA-modified ApoB100 protein and oxidized lipid. At that time, when the oxidized oxidized LDL (MDA-LDL) is digested with a protease having cleavage site specificity, for example, trypsin digestion, the MDA-modified ApoB100 protein is fragmented into peptides. The group of peptide fragments that are produced are eluted in the liquid phase. Along with the fragmentation of the MDA-modified ApoB100 protein, a group of peptide fragments eluted in the liquid phase is also separated from the lipid component. When the liquid phase and the beads are separated by solid-liquid separation, and the liquid phase is recovered, a group of peptide fragments generated by enzymatic digestion of the MDA-modified ApoB100 protein is recovered in the recovered liquid phase. Since the ApoB100 protein itself is a huge protein consisting of 4563 amino acid residues, the total number of a group of peptide fragments generated by trypsin digestion of the ApoB100 protein exceeds 400 types.

Therefore, after separating all of a group of peptide fragments, whether or not there is an MDA-modified Lys residue (MDA-ized site) or an oxidized Met residue (oxidized site) in each peptide fragment. Although it is not impossible in principle, it is technically difficult to determine. Considering this point, when a group of more than 400 kinds of peptide fragments are fractionated into a plurality of groups based on their isoelectric points (pI), each fractionated group is divided into about 20 kinds of peptide fragments. It was found that it can be a group. In addition, about 20 kinds of peptide fragments constituting each group have similar isoelectric points (pI), but accidentally, two kinds of peptide fragments having the same molecular weight (M _peptide ) are the same. I thought that there was almost no probability of being included in the group.

Therefore, when the molecular weight (M _peptide ) of a peptide fragment is measured using mass spectrometry for about 20 types of peptide fragments that have similar isoelectric points (pI) and constitute each group, It was found that the peptide fragment can be detected as a peptide fragment of (pI, M _peptide ).

  In that case, the amino acid sequence of each peptide fragment is a partial amino acid sequence in the amino acid sequence of ApoB100 protein. For example, in the trypsin digestion treatment, the peptide chain is cleaved at the C-terminal side of the Arg residue or Lys residue, so the amino acid residue at the C-terminus of each peptide fragment is an Arg residue or Lys residue, The N-terminal amino acid residue of each peptide fragment is an amino acid residue located on the C-terminal side of the Arg residue or Lys residue in the amino acid sequence of the ApoB100 protein.

A partial amino acid sequence satisfying the above conditions is selected from the amino acid sequence of the ApoB100 protein, and a group of “presumed peptide fragments” consisting of the partial amino acid sequences is prepared. The molecular weight estimated from (M _{predicted-peptide} ) is calculated. Measured (pI, M _peptide) molecular weight of the peptide fragment of (M _peptide), consistent with the measurement error range, the molecular weight deduced from the "partial amino acid sequence" (M _{predicted-peptide)} are found, measured It was found that the amino acid sequence of the obtained (pI, M _peptide ) peptide fragment can be estimated with high accuracy to be its “partial amino acid sequence”.

On the other hand, when the measured peptide fragment of (pI, M _peptide ) contains an MDA-modified Lys residue (MDA-modified site) or an oxidized Met residue (oxidized site), the measured molecular weight (M _peptide ) and measurement It was conceived that there was almost no probability of finding a molecular weight (M _{predicted-peptide} ) estimated from the “partial amino acid sequence” that matched within the error range. In other words, among the measured peptide fragments of (pI, M _peptide ), the molecular weight estimated from the “partial amino acid sequence” that matches the measured molecular weight (M _peptide ) within the measurement error range (M _predicted- It was conceived that a _peptide not found) may contain an MDA-modified Lys residue (MDA-ized site) or an oxidized Met residue (oxidized site) in its amino acid sequence. Of the measured peptide fragments of (pI, M _peptide ), the molecular weight (M _{predicted-peptide} ) estimated from the “partial amino acid sequence” that matches the measured molecular weight (M _peptide ) within the measurement error range is Those not found were thought to be likely to contain at least some modified amino acid residues in the amino acid sequence.

  Depending on the level of oxidative stress, the distribution of the degree of oxidation in the resulting mixture of oxidized LDL is different. Due to the difference in the distribution of the degree of oxidation, there is a difference in the distribution of the total number of MDA-modified Lys residues in the ApoB100 protein in the generated oxidized LDL and the distribution of the total number of oxidized Met residues. is expected.

Therefore, depending on the level of oxidative stress, MDA Lys residues or oxidized Met residues in the amino acid sequence in oxidized LDL, particularly in a group of peptide fragments derived from ApoB100 protein in MDA-LDL. content level C (pI, M _peptide) peptide fragments containing a group (pI, M _peptide) are also expected to change. For example, the ratio of the content concentration C _{MDA-LDL of MDA-LDL} to the content concentration C (pI, M _peptide ) of the peptide fragment (pI, M _peptide ) containing _MDA-modified Lys residue or oxidized Met residue, (C (pI, M _peptide ) / C _MDA-LDL ) is predicted to vary depending on the level of oxidative stress. In this case, a peptide fragment (pI, M _peptide containing MDA-modified Lys residue or oxidized Met residue, which is commonly found in ApoB100 protein in MDA-LDL, regardless of the level of oxidative stress. ), (C (pI, M _peptide ) / C _MDA-LDL ) is expected to have little variation due to the level of oxidative stress. On the other hand, depending on the level of oxidative stress, the peptide fragment containing MDA-modified Lys residue or oxidized Met residue (pI, M) whose probability of being found in ApoB100 protein in MDA-LDL varies greatly. _In ( _peptide ), (C (pI, M _peptide ) / C _MDA-LDL ) is expected to vary greatly due to the level of oxidative stress.

In view of the above-mentioned expectation, the present inventors estimated that it is possible that the amino acid sequence of the ApoB100 protein in MDA-LDL contains an MDA-modified Lys residue or an oxidized Met residue ( A group of pI, M _peptide ) is selected, and among the group of peptide fragments (pI, M _peptide ) presumed to contain MDA-modified Lys residue or oxidized Met residue, (C (pI , M _peptide ) / C _MDA-LDL ), we proceeded to select subgroups in which fluctuations due to the level of oxidative stress level exceeded a certain level.

At that time, focusing on the fact that one particle of MDA-LDL, which is oxidized LDL, contains only one molecule of ApoB100 protein, instead of measuring the _MDA-LDL content concentration C _MDA-LDL , MDA the content level C _{MDA-LDL-ApoB100} of ApoB100 protein molecules in -LDL, the ratio between the content concentration C (pI, M _peptide) peptide fragments _{(pI, M peptide), (} C (pI, M peptide) / C _{MDA-LDL-ApoB100} ) can be regarded as (C (pI, M _peptide ) / C _{MDA-LDL-ApoB100} ) = (C (pI, M _peptide ) / C _MDA-LDL ). I found out.

Is oxidized LDL, generated by enzymatic digestion of ApoB100 protein in MDA-LDL, the average value of the content level C (pI, M _peptide) of a group of peptide fragments of the total number of types _{N peptide (pI, M peptide)} , C (PI, M _peptide ) _av. = (ΣC (pI, M _peptide )) / N _peptide is almost equal to the concentration of ApoB100 protein molecule C _{MDA-LDL-ApoB100 in MDA-LDL} , that is, C (pI , M _peptide ) _av. ≒ C _{MDA-LDL-ApoB100} .

In addition, by isoelectric focusing, after grouping based on the isoelectric point (pI), from the group of peptide fragments constituting each group, the peptide fragments of (pI, M _peptide ) are separated by mass spectrometry. is the inclusion of a peptide fragment of (pI, M _peptide) ionic species derived from the peptide fragments of [M _peptide ⁺ H] + peak intensity _{I (pI, [M peptide +} H] +) , said (pI, M _peptide) It is proportional to the concentration C (pI, M _peptide ). (PI, M _peptide) from the peptide fragment, considering the ionic species derived from the peptide fragment [M _peptide ⁺ H] + generation efficiency (the ionization _{efficiency) p (pI, M peptide)} , I (pI, [M peptide + H ] ⁺ ) ≈p (pI, M _peptide ) · C (pI, M _peptide ).

For a group of peptide fragments (pI, M _peptide ) of the total number N _peptide , the measured ionic species [M _peptide + H] ⁺ derived from the peptide fragment of the (pI, M _peptide ) I (pI, [M _peptide + H] ⁺ ) / p (pI, M _peptide ) ≈C (pI, M _peptide ) average value, {I (pI, [M _peptide + H] ⁺ ) / p (pI, M _peptide )} _av. = (Σ {I (pI, [M _peptide + H] ⁺ )) / p (pI, M _peptide )} / N _peptide ≈ (ΣC (pI, M _peptide )) / N _peptide contains the ApoB100 protein molecule in MDA-LDL Concentration is proportional to _{CMDA-LDL-ApoB100} . That, {I (pI, [M peptide + H] +) / p (pI, M peptide)} av. Proportionality αC _{MDA-LDL-ApoB100} holds.

Furthermore, the measured said (pI, M _peptide) ionic species derived from the peptide fragments of [M _peptide ⁺ H] + peak intensity _{I (pI, [M peptide +} H] +) and, {I (pI, [M peptide + H] ⁺ ) / p (pI, M _peptide )} _av. Ratio; I (pI, [M _peptide + H] ⁺ ) / {I (pI, [M _peptide + H] ⁺ ) / p (pI, M _peptide ) } _Av. Is proportional to (C (pI, M _peptide ) / C _{MDA-LDL-ApoB100} ). That is, I (pI, [M _peptide + H] ⁺ ) / {I (pI, [M _peptide + H] ⁺ ) / p (pI, M _peptide )} _av. ∝ (C (pI, M _peptide ) / C _{MDA LDL-ApoB100} ) proportional relationship holds.

Using the above proportional relationship, the present inventors specify from the peak position m / Z = (M _peptide +1) / 1 of the ion species [M _peptide + H] ⁺ derived from the peptide fragment of (pI, M _peptide ). Based on the molecular weight (M _peptide ), a group of _peptide fragments (pI, M _peptide ) presumed to contain MDA-modified Lys residues or oxidized Met residues in the amino acid sequence is selected, Furthermore, among the group of peptide fragments (pI, M _peptide ) presumed to contain MDA-modified Lys residues or oxidized Met residues, I (pI, [M _peptide + H] ⁺ ) / I (pI , [M _peptide + H] ⁺ ) _av . I (pI, [M _peptide + H] ⁺ ) / I (pI, [M _peptide + H] ⁺ ) _av. Selection considering the proportional relationship of C ( _p (M, _peptide ) / _{CMDA-LDL-ApoB100} ) The subgroup to be processed is substantially equivalent to the subgroup in which the variation due to the level of oxidative stress in (C (pI, M _peptide ) / C _MDA-LDL ) exceeds a certain level.

In particular, when the Matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS) method is adopted as the mass spectrometry, the C-terminal amino acid residue obtained by the trypsin digestion process is the Arg residue or The generation efficiency (ionization efficiency) p (pI, M _peptide ) of the ionic species [M _peptide + H] ⁺ derived from a peptide fragment that is a Lys residue is relatively high. Therefore, it was confirmed that the measurement accuracy of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the measured ion species [M _peptide + H] ⁺ derived from the peptide fragment of the (pI, M _peptide ) was also increased.

In addition, since the peptide fragment obtained by trypsin digestion is enzymatically digested at the C-terminal side of the Arg residue or Lys residue, in principle, other than the Arg residue or Lys residue at the C-terminus Has no basic amino acid residues. Accordingly, the isoelectric point pI of the peptide fragment obtained by trypsin digestion generally depends on one of the basic amino acids Arg or Lys and the total number of acidic amino acids Asp and Glu contained therein. Using this feature, it is divided into a subgroup in which the total number of contained acidic amino acids is 0, a subgroup in which the total number of contained acidic amino acids is 1, and a subgroup in which the total number of contained acidic amino acids is 2 or more. Each subgroup was confirmed to be separable based on pI. That is, when the molecular weights (M _peptide ) of the peptide fragments coincide, if the total number of acidic amino acids contained in the peptide fragments is different, the pI shows a significant difference. It was confirmed that pI, M _peptide ) can be separated in advance by isoelectric focusing.

  In addition, when the amino acid sequence contains an MDA-modified Lys residue, the probability of enzymatic digestion at the C-terminal side of the MDA-modified Lys residue when subjected to trypsin digestion is the residual Lys residue that has not undergone MDA conversion. It was also confirmed that it was significantly lower than the probability of enzymatic digestion on the C-terminal side of the group. Therefore, it is possible to obtain a specific peptide fragment containing MDA-modified Lys residue in a shape in which two consecutive peptide fragments are linked without undergoing enzymatic digestion on the C-terminal side of the MDA-modified Lys residue. confirmed. That is, it was also confirmed that the concentration of a specific peptide fragment containing MDA-modified Lys residue in the shape in which two continuous peptide fragments were linked relatively increased with the progress of MDA conversion.

  On the other hand, when the Lys residue is not subjected to MDA conversion, the probability that the Lys residue is not enzymatically digested at the C-terminal side of the Lys residue is generally low, so the concentration of the peptide fragment in a form in which two consecutive peptide fragments are linked is , Usually stays at a low level. Also included are peptide fragments having a shape in which two consecutive peptide fragments are linked, and specific peptide fragments having a shape in which two consecutive peptide fragments are linked and containing an MDA-modified Lys residue. Although the total number of acidic amino acids is the same, there is a difference in the number of basic amino acids, so their isoelectric points (pI) are clearly different. Therefore, these two peptide fragments can be separated in advance by isoelectric focusing.

  At the same time, it was also confirmed that the concentration of peptide fragments generated by enzymatic digestion on the C-terminal side of the MDA-modified Lys residue relatively increased with the progress of MDA-ization. However, when trypsin digestion treatment is performed, the probability of enzymatic digestion at the C-terminal side of the MDA-modified Lys residue is generally low, so a peptide generated by undergoing enzymatic digestion at the C-terminal side of the MDA-modified Lys residue Fragment concentration usually remains at a low level. However, when the progress of MDA conversion becomes significant, the concentration of peptide fragments produced by enzymatic digestion on the C-terminal side of the MDA-modified Lys residue reaches a considerable level. The peptide fragment produced by enzymatic digestion on the C-terminal side of the MDA-modified Lys residue has the same total number of acidic amino acids as the peptide fragment that does not have the MDA-modified Lys residue and has the same amino acid sequence However, instead of the Lys residue, it has an MDA-modified Lys residue, and its isoelectric point (pI) is significantly shifted to the acidic side. Therefore, these two peptide fragments can be separated in advance by isoelectric focusing.

  A peptide fragment containing an oxidized Met residue and a peptide fragment not containing an oxidized Met residue and having the same amino acid sequence show similar isoelectric points (pI) because the total number of acidic amino acids contained is equal. Of course, since the masses of oxidized Met residues and non-oxidized Met residues are different, it is possible to distinguish these two peptide fragments by mass spectrometry. As the oxidation of Met residues proceeds, the concentration of peptide fragments containing oxidized Met residues increases, while the concentration of peptide fragments that do not contain oxidized Met residues and have the same amino acid sequence decreases. An increase in the concentration of a peptide fragment containing an oxidized Met residue is an index reflecting the progress of oxidation of the Met residue.

Considering the above points, among the peptide fragments containing the MDA-modified Lys residue or oxidized Met residue (pI, M _peptide ) in the amino acid sequence, in particular, the peptide fragment containing the oxidized Met residue, Based on a change in concentration of a specific peptide fragment containing an MDA-modified Lys residue in the form of two peptide fragments linked, and a peptide fragment generated by enzymatic digestion at the C-terminal side of the MDA-modified Lys residue Therefore, it was determined that it was possible to estimate the level of oxidative stress.

  After the above examination, among the ApoB100 proteins contained in LDL contained in human blood, oxidized LDL, in particular, MDA-lysed Lys residues present in ApoB100 protein contained in MDA-LDL , And Met residues that are MDA-modified Lys residues in which the probability of undergoing MDA is clearly changed due to a difference in the level of oxidative stress, and those in which the probability of undergoing oxidation is clearly changed The oxidized Met residues to be selected were selected.

  Instead of “level of oxidative stress”, blood was collected from a group of patients whose coronary artery lesion branch number was determined to be the progression of ischemic heart disease, ie, the number of coronary artery lesion branches (ACI-TIPI score) Blood samples collected from a group of healthy individuals determined to have 0 coronary lesions are regarded as blood samples with a high level of oxidative stress and blood samples with a low level of oxidative stress, respectively. . Contained in MDA-LDL, contained in MDA-LDL, contained in blood samples with "high oxidative stress" and contained in MDA-LDL, contained in blood samples with "low oxidative stress" When comparing ApoB100 proteins, the MDA-modified Lys residue (MDA-ized site) having a clearly different probability of undergoing MDA conversion, and the oxidized Met residue having a clearly different probability of undergoing oxidation Sorting was performed.

At that time, the modification of the Lys residue side chain amino group (—NH ₂ ) by MDA is a modification involving one molecule of MDA, which is the MDA conversion of the Lys residue (—NH—CH═CH—CHO). In addition to the above, it was confirmed that DHP (dihydroxypyridine) of the Lys residue corresponding to the modification involving multiple MDA molecules was also present. The DHP conversion of the Lys residue corresponds to a modification involving multiple MDA molecules, and is assumed to occur when the “oxidative stress level” is high. Therefore, the ApoB100 protein contained in the MDA-LDL contained in the blood sample with “high oxidative stress” and the MDA-LDL contained in the blood sample with “low oxidative stress” When the ApoB100 protein contained in was compared, DHP-modified Lys residues (DHP-ized sites) that clearly differ in the probability of undergoing DHP were also selected.

  As a result of the above selection, the present inventors have included the ApoB100 protein contained in the MDA-LDL, which is contained in the blood sample having “high oxidative stress”, and the blood sample having “low oxidative stress”. When the ApoB100 protein contained in the MDA-LDL contained in the MDA-LDL is compared, the probability of undergoing MDA conversion is clearly different. As the DHP-modified Lys residue (DHP-ized site) that is clearly different, and the oxidized Met residue (oxidized site) that is clearly different in the probability of being oxidized, the following table is included in the amino acid sequence of the ApoB100 protein. 1-1, Lys residues at amino acid residue positions shown in Table 1-2, and Met residues were selected.

  That is, when evaluating the oxidation of amino acid residues in the ApoB100 protein contained in MDA-LDL and the degree of progress of modification by MDA (progression of oxidation), it is contained in the collected MDA-LDL. Among the ApoB100 proteins, Lys residues at amino acid residue positions shown in Table 1-1 and Table 1-2, and Met residues are modified to MDA-modified Lys residues, DHP-modified Lys residues, or Based on the ratio of oxidation to oxidized Met residues, it was found that the “degree of oxidation progress” can be evaluated with high accuracy.

  Since the oxidation of amino acid residues in the ApoB100 protein contained in MDA-LDL and the degree of progress of modification by MDA (the degree of oxidation progress) depend on the “level of oxidative stress”, MDA-LDL Based on the evaluation result of “progression of oxidation” in the ApoB100 protein contained therein, it was found that the level of “oxidative stress level” can be estimated with high reliability.

  Furthermore, the level of oxidative stress is related to the progression of atherosclerosis in the coronary arteries, and the result of reliable estimation of the level of the level of oxidative stress is the progression of atherosclerosis in the coronary arteries. It can be used as one of the indices reflecting the pathological condition when evaluating the degree of the disease. That is, when evaluating the degree of progression of atherosclerosis in the coronary artery, the evaluation result of “degree of oxidation” in the ApoB100 protein contained in MDA-LDL reflects the pathology derived from “oxidative stress”. We found that it can be used as one of the indicators. In other words, “a peptide fragment derived from ApoB100 protein contained in MDA-LDL” that can be used for evaluation of “degree of oxidation” in ApoB100 protein contained in MDA-LDL is a heart disease. The present inventors have found that the present invention can be used as a biomarker that reflects the pathological condition of the disease, specifically, a biomarker that reflects the degree of progression of atherosclerosis in the coronary artery.

  Based on the series of findings described above, the present inventors have completed the present invention.

  First, the first aspect of the present invention is the oxidation of amino acid residues in the ApoB100 protein contained in the MDA-LDL collected from a blood sample collected from a subject, and the progress of modification by MDA. It is an invention of a method for evaluating the degree (progress of oxidation).

The method for evaluating the progress of oxidation in the ApoB100 protein contained in the MDA-LDL according to the first aspect of the first aspect of the present invention,
A method for evaluating the progress of oxidation in ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject,
It has the following steps (Step 1) to (Step 6),
(Process 1)
Applying MDA-LDL from a blood sample collected from a subject by applying an immunoprecipitation method using an anti-MDA-LDL monoclonal antibody;
(Process 2)
A step of allowing trypsin to act on the ApoB100 protein contained in the fractionated MDA-LDL to prepare a group of peptide fragments derived from the ApoB100 protein that have been digested with trypsin;
(Process 3)
A group of peptide fragments derived from the ApoB100 protein digested with trypsin by applying isoelectric focusing method is fractionated into predetermined isoelectric points according to the isoelectric point of each peptide fragment. Separating into a plurality of peptide fragment subgroups fractionated based on points;
(Process 4)
About the partial group of a plurality of peptide fragments fractionated based on the isoelectric point, the mass of the peptide fragment (M _peptide ) contained in the partial group of each peptide fragment is used as a mass analysis means, and MALDI-TOF The peptide fragment contained in each peptide fragment subgroup was measured based on the isoelectric point (pI) and mass (M _peptide ), and the peptide fragment (pI, M _peptide ), and measuring the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from each peptide fragment (pI, M _peptide );
(Process 5)
From a group of identified peptide fragments (pI, M _peptide ), from the Lys residue at the amino acid residue position shown in Table 1-1 below, and the Met residue at the amino acid residue position shown in Table 1-2 At least one oxidized peptide fragment corresponding to a peptide fragment derived from the ApoB100 protein, comprising at least one Lys residue or Met residue selected from the group, wherein the Lys residue or Met residue is oxidized. Selecting one process;

(Step 6)
Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the selected oxidized peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured , Evaluating the progress of oxidation in the ApoB100 protein contained in the fractionated MDA-LDL;
In step 6, the evaluation of the progress of oxidation is as follows:
(Sub-step 6-1)
Identified peptide fragments (pI, M _peptide) the total number of kinds of peptide fragments contained in the group of the N _{total-peptide,} are included in the group of identified peptide fragments (pI, M _peptide), the total number N _Total- Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the peptide fragment of the _peptide type, the peak intensity I (pI, [M _peptide + H] ⁺ ) average value, I (pI, [M _peptide + H] ⁺ ) _av calculating step;
(Sub-step 6-2)
The measured value of peak intensity I (pI, [M _peptide + H] ⁺ ) of ionic species [M _peptide + H] ⁺ derived from the selected oxidized peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured and calculated ApoB100 protein contained in the fractionated MDA-LDL by comparing the average value of the measured peak intensity I (pI, [M _peptide + H] ⁺ ), I (pI, [M _peptide + H] ⁺ ) _av In which a content ratio of oxidized ApoB100 protein containing the selected oxidized peptide fragment is estimated;
(Sub-step 6-3)
The higher the content ratio of the oxidized ApoB100 protein that contains the selected oxidized peptide fragment in the ApoB100 protein contained in the fractionated MDA-LDL, the more the ApoB100 protein contained in the fractionated MDA-LDL. The step of evaluating that the degree of progress of oxidation is high;
The evaluation method is characterized in that the progress of oxidation is evaluated based on the above-mentioned steps (Sub-Step 6-1) to (Sub-Step 6-3).

The method for evaluating the progress of oxidation in the ApoB100 protein contained in the MDA-LDL according to the second aspect of the first aspect of the present invention,
A method for evaluating the progress of oxidation in ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject,
It has the following steps (Step 1) to (Step 6),
(Process 1)
Applying MDA-LDL from a blood sample collected from a subject by applying an immunoprecipitation method using an anti-MDA-LDL monoclonal antibody;
(Process 2)
A step of allowing trypsin to act on the ApoB100 protein contained in the fractionated MDA-LDL to prepare a group of peptide fragments derived from the ApoB100 protein that have been digested with trypsin;
(Process 3)
A group of peptide fragments derived from the ApoB100 protein digested with trypsin by applying isoelectric focusing method is fractionated into predetermined isoelectric points according to the isoelectric point of each peptide fragment. Separating into a plurality of peptide fragment subgroups fractionated based on points;
(Process 4)
About the partial group of a plurality of peptide fragments fractionated based on the isoelectric point, the mass of the peptide fragment (M _peptide ) contained in the partial group of each peptide fragment is used as a mass analysis means, and MALDI-TOF The peptide fragment contained in each peptide fragment subgroup was measured based on the isoelectric point (pI) and mass (M _peptide ), and the peptide fragment (pI, M _peptide ), and measuring the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from each peptide fragment (pI, M _peptide );
(Process 5)
Selecting at least one peptide fragment selected from the group consisting of peptide fragments containing the amino acid residues shown in Table 2 below, from a group of identified peptide fragments (pI, M _peptide );

(Step 6)
Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the selected peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured , Assessing the degree of oxidation in the ApoB100 protein contained in the MDA-LDL taken;
In step 6, the evaluation of the progress of oxidation is as follows:
(Sub-step 6-1)
Identified peptide fragments (pI, M _peptide) the total number of kinds of peptide fragments contained in the group of the N _{total-peptide,} are included in the group of identified peptide fragments (pI, M _peptide), the total number N _Total- Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the peptide fragment of the _peptide type, the peak intensity I (pI, [M _peptide + H] ⁺ ) average value, I (pI, [M _peptide + H] ⁺ ) _av calculating step;
(Sub-step 6-2)
Measured value of peak intensity I (pI, [M _peptide + H] ⁺ ) of ionic species [M _peptide + H] ⁺ derived from the selected peptide fragment, calculated as I (pI, [M _peptide + H] ⁺ ) _measured In the ApoB100 protein contained in the fractionated MDA-LDL, the average value of the peak intensity I (pI, [M _peptide + H] ⁺ ) and I (pI, [M _peptide + H] ⁺ ) _av are compared. Estimating the content of oxidized ApoB100 protein that produces the selected peptide fragment by trypsin digestion;
(Sub-step 6-3)
The higher the content ratio of the oxidized ApoB100 protein that is produced by trypsin digestion of the selected peptide fragment in the ApoB100 protein contained in the fractionated MDA-LDL, the more it is contained in the fractionated MDA-LDL. A step of evaluating that the degree of oxidation is high in the ApoB100 protein;
The evaluation method is characterized in that the progress of oxidation is evaluated based on the above-mentioned steps (Sub-Step 6-1) to (Sub-Step 6-3).

The method for evaluating the progress of oxidation in the ApoB100 protein contained in the MDA-LDL according to the third aspect of the first aspect of the present invention,
A method for evaluating the progress of oxidation in ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject,
It has the following steps (Step 1) to (Step 6),
(Process 1)
Applying MDA-LDL from a blood sample collected from a subject by applying an immunoprecipitation method using an anti-MDA-LDL monoclonal antibody;
(Process 2)
A step of allowing trypsin to act on the ApoB100 protein contained in the fractionated MDA-LDL to prepare a group of peptide fragments derived from the ApoB100 protein that have been digested with trypsin;
(Process 3)
A group of peptide fragments derived from the ApoB100 protein digested with trypsin by applying isoelectric focusing method is fractionated into predetermined isoelectric points according to the isoelectric point of each peptide fragment. Separating into a plurality of peptide fragment subgroups fractionated based on points;
(Process 4)
About the partial group of a plurality of peptide fragments fractionated based on the isoelectric point, the mass of the peptide fragment (M _peptide ) contained in the partial group of each peptide fragment is used as a mass analysis means, and MALDI-TOF The peptide fragment contained in each peptide fragment subgroup was measured based on the isoelectric point (pI) and mass (M _peptide ), and the peptide fragment (pI, M _peptide ), and measuring the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from each peptide fragment (pI, M _peptide );
(Process 5)
From among a group of identified peptide fragments (pI, M _peptide ), a peptide fragment containing the MDA-modified Lys residue shown in Table 3 below, and a peptide containing the DHP-modified Lys residue shown in Table 4 inside At least a Lys residue or Met selected from the group consisting of a fragment, a peptide fragment containing the MDA-modified Lys residue shown in Table 5 at its C-terminus, and a peptide fragment containing an oxidized Met residue shown in Table 6 Selecting at least one oxidized peptide fragment corresponding to the peptide fragment whose residue has undergone oxidation;

(Step 6)
Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the selected oxidized peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured , Evaluating the progress of oxidation in the ApoB100 protein contained in the fractionated MDA-LDL;
In step 6, the evaluation of the progress of oxidation is as follows:
(Sub-step 6-1)
Identified peptide fragments (pI, M _peptide) the total number of kinds of peptide fragments contained in the group of the N _{total-peptide,} are included in the group of identified peptide fragments (pI, M _peptide), the total number N _Total- Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the peptide fragment of the _peptide type, the peak intensity I (pI, [M _peptide + H] ⁺ ) average value, I (pI, [M _peptide + H] ⁺ ) _av calculating step;
(Sub-step 6-2)
The measured value of peak intensity I (pI, [M _peptide + H] ⁺ ) of ionic species [M _peptide + H] ⁺ derived from the selected oxidized peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured and calculated ApoB100 protein contained in the fractionated MDA-LDL by comparing the average value of the measured peak intensity I (pI, [M _peptide + H] ⁺ ), I (pI, [M _peptide + H] ⁺ ) _av In which a content ratio of oxidized ApoB100 protein containing the selected oxidized peptide fragment is estimated;
(Sub-step 6-3)
The higher the content ratio of the oxidized ApoB100 protein that contains the selected oxidized peptide fragment in the ApoB100 protein contained in the fractionated MDA-LDL, the more the ApoB100 protein contained in the fractionated MDA-LDL. The step of evaluating that the degree of progress of oxidation is high;
The evaluation method is characterized in that the progress of oxidation is evaluated based on the above-mentioned steps (Sub-Step 6-1) to (Sub-Step 6-3).

In the method for evaluating the progress of oxidation in the ApoB100 protein contained in the MDA-LDL according to the third aspect of the first aspect of the present invention,
In step 5, at least one of the oxidized peptide fragments selected is
It is preferable to select from a group of peptide fragments containing the MDA-modified Lys residue shown in Table 3 therein.

In the evaluation method of the first aspect of the present invention,
The anti-MDA-LDL monoclonal antibody used for fractionation of MDA-LDL by immunoprecipitation in Step 1 is a monoclonal antibody specific for MDA-converted ApoB100 protein contained in MDA-LDL. It is preferable.

  Further, the fractionation of peptide fragments to which the isoelectric focusing method is applied in the step 3 can be carried out using an isoelectric focusing protein separation chip.

  Next, according to the second aspect of the present invention, the degree of progression of the oxidation of amino acid residues in the ApoB100 protein contained in MDA-LDL and the modification by MDA according to the first aspect of the present invention described above. (Evaluation of “progression of oxidation”), “progress of oxidation in ApoB100 protein contained in MDA-LDL” was evaluated, and based on the evaluation result of “progress of oxidation” This is an invention of a method for estimating the level of “oxidative stress level” in the process in which sorted MDA-LDL is generated from LDL.

The method for estimating the level of “oxidative stress level” in the process of producing MDA-LDL from LDL according to the first aspect of the second aspect of the present invention,
Oxidative stress in the process of generating MDA-LDL from LDL based on the evaluation results of the degree of oxidation of ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject A method for estimating the level of
Includes the following process A and process B (process A)
MDA according to the first aspect of the first aspect of the present invention was used to evaluate the progress of oxidation in the ApoB100 protein contained in the MDA-LDL fractionated from the blood sample collected from the subject. -A step performed using a method for evaluating the progress of oxidation in the ApoB100 protein contained in LDL;
(Process B)
In the process in which the MDA-LDL is produced from LDL based on the evaluation result of the degree of oxidation of ApoB100 protein contained in the MDA-LDL to be collected from the blood sample collected from the subject, Estimating the level of oxidative stress in an environment in which MDA-LDL production has progressed;
In Step B, the estimation of the level of oxidative stress is
Oxidation of the environment in which the production of MDA-LDL has progressed in the process of producing MDA-LDL from LDL the higher the degree of oxidation in ApoB100 protein contained in the fractionated MDA-LDL It is an estimation method characterized by estimating that the level of stress is high.

The method for estimating the level of “oxidative stress level” in the process of producing MDA-LDL from LDL according to the second aspect of the second aspect of the present invention,
Oxidative stress in the process of generating MDA-LDL from LDL based on the evaluation results of the degree of oxidation of ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject A method for estimating the level of
Includes the following process A and process B (process A)
MDA according to the second aspect of the first aspect of the present invention was used to evaluate the progress of oxidation in the ApoB100 protein contained in the MDA-LDL fractionated from the blood sample collected from the subject. -A step performed using a method for evaluating the progress of oxidation in the ApoB100 protein contained in LDL;
(Process B)
In the process in which the MDA-LDL is produced from LDL based on the evaluation result of the degree of oxidation of ApoB100 protein contained in the MDA-LDL to be collected from the blood sample collected from the subject, Estimating the level of oxidative stress in an environment in which MDA-LDL production has progressed;
In Step B, the estimation of the level of oxidative stress is
Oxidation of the environment in which the production of MDA-LDL has progressed in the process of producing MDA-LDL from LDL the higher the degree of oxidation in ApoB100 protein contained in the fractionated MDA-LDL It is an estimation method characterized by estimating that the level of stress is high.

The method for estimating the level of “oxidative stress level” in the process of producing MDA-LDL from LDL according to the third aspect of the second aspect of the present invention,
Oxidative stress in the process of generating MDA-LDL from LDL based on the evaluation results of the degree of oxidation of ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject A method for estimating the level of
Includes the following process A and process B (process A)
MDA according to the third aspect of the first aspect of the present invention was used to evaluate the progress of oxidation in the ApoB100 protein contained in the MDA-LDL collected from the blood sample collected from the subject. -A step performed using a method for evaluating the progress of oxidation in the ApoB100 protein contained in LDL;
(Process B)
In the process in which the MDA-LDL is produced from LDL based on the evaluation result of the degree of oxidation of ApoB100 protein contained in the MDA-LDL to be collected from the blood sample collected from the subject, Estimating the level of oxidative stress in an environment in which MDA-LDL production has progressed;
In Step B, the estimation of the level of oxidative stress is
Oxidation of the environment in which the production of MDA-LDL has progressed in the process of producing MDA-LDL from LDL the higher the degree of oxidation in ApoB100 protein contained in the fractionated MDA-LDL It is an estimation method characterized by estimating that the level of stress is high.

  As a third aspect of the present invention, when evaluating the degree of progression of atherosclerosis in the coronary artery, which is a major factor of heart disease, particularly ischemic heart disease, This is a method of using the degree of oxidation of ApoB100 protein contained in MDA-LDL to be collected from a blood sample collected from a subject. Specifically, prior to the evaluation of the degree of progression of atherosclerosis in the coronary arteries, in order to grasp the pathological condition of the subject, from the blood sample collected from the subject, during the MDA-LDL to be sorted It is an invention of a method for selecting an oxidized peptide fragment derived from the ApoB100 protein, which is suitable for evaluation of the progress of oxidation, as a marker used for the evaluation when evaluating the progress of oxidation in the included ApoB100 protein.

  Accordingly, the third aspect of the present invention is an invention corresponding to “a method for selecting a diagnostic marker that can be used for heart disease diagnosis”, and more specifically, prior to the diagnosis of heart disease, the condition of the subject is grasped. When evaluating the progress of oxidation in the ApoB100 protein contained in the MDA-LDL collected from a blood sample collected from a subject, the marker used for the evaluation is It is an invention of a method for selecting an oxidized peptide fragment derived from the ApoB100 protein, which is suitable for evaluation of the progress of oxidation.

According to the first aspect of the third aspect of the present invention, "a method for selecting a diagnostic marker that can be used for diagnosis of heart disease"
Prior to the diagnosis of heart disease, the progress of oxidation in the ApoB100 protein contained in the MDA-LDL sampled from the blood sample collected from the subject is evaluated for the purpose of grasping the condition of the subject. In this case, as a marker used for the evaluation, a method for selecting an oxidized peptide fragment derived from the ApoB100 protein, which is suitable for the evaluation of the progress of the oxidation,
As a marker used for the evaluation,
At least one Lys residue or Met residue selected from the group consisting of the Lys residue at the amino acid residue position shown in Table 7-1 below and the Met residue at the amino acid residue position shown in Table 7-2 Selecting at least one oxidized peptide fragment corresponding to a peptide fragment derived from ApoB100 protein containing a group, wherein the Lys residue or Met residue is oxidized

This is a method for selecting a marker.

According to the second aspect of the third aspect of the present invention, "a method for selecting a diagnostic marker that can be used for diagnosis of heart disease"
Prior to the diagnosis of heart disease, the progress of oxidation in the ApoB100 protein contained in the MDA-LDL sampled from the blood sample collected from the subject is evaluated for the purpose of grasping the condition of the subject. In this case, as a marker to be used for the evaluation, a method for selecting a peptide fragment derived from the ApoB100 protein suitable for the evaluation of the progress of the oxidation,
As a marker used for the evaluation,
Select at least one peptide fragment selected from the group consisting of peptide fragments containing amino acid residues shown in Table 8 below.

This is a method for selecting a marker.

According to the third aspect of the third aspect of the present invention, the “method for selecting a diagnostic marker that can be used for heart disease diagnosis”
Prior to the diagnosis of heart disease, the progress of oxidation in the ApoB100 protein contained in the MDA-LDL sampled from the blood sample collected from the subject is evaluated for the purpose of grasping the condition of the subject. In this case, as a marker used for the evaluation, a method for selecting an oxidized peptide fragment derived from the ApoB100 protein, which is suitable for the evaluation of the progress of the oxidation,
As a marker used for the evaluation,
A peptide fragment containing the MDA-modified Lys residue shown in Table 9 below, a peptide fragment containing the DHP-modified Lys residue shown in Table 10 inside, and the MDA-modified Lys residue shown in Table 11 at the C-terminus An oxidized peptide fragment corresponding to a peptide fragment selected from the group consisting of peptide fragments containing oxidized Met residues shown in Table 12 and at least Lys residues or Met residues undergoing oxidation Select at least one

This is a method for selecting a marker.

In the “method for selecting a diagnostic marker that can be used for heart disease diagnosis” according to the third aspect of the first aspect of the present invention,
At least one of the selected oxidized peptide fragments is
It is preferable to select from a group of peptide fragments containing the MDA-modified Lys residue shown in Table 9 therein.

  According to the method of the present invention, it is possible to quickly and easily detect a biomarker related to the pathological condition of heart disease from a sample that is relatively free of mental and physical burdens on patients such as serum and plasma.

A group of oxidized peptide fragments that can be used as a “diagnostic marker” in the “method for selecting a diagnostic marker that can be used for heart disease diagnosis” according to the third aspect of the present invention, that is, from a blood sample collected from a subject, Adopted in the process of searching for a group of oxidized peptide fragments derived from ApoB100 protein suitable for evaluation of the progress of oxidation when evaluating the progress of oxidation in ApoB100 protein contained in sorted MDA-LDL It is the figure which shows typically the step structure of the search process. FIG. 1 schematically shows a configuration of a series of sub-steps adopted when performing the step of “sample pretreatment” 101 in the step configuration of the search process for a group of oxidized peptide fragments that can be used as “diagnostic markers”. FIG. The “isoelectric focusing protein separation chip” employed when performing the step of “isoelectric focusing” 102 in the step configuration of the search process for a group of oxidized peptide fragments that can be used as “diagnostic markers” shown in FIG. FIG. In the step configuration of the search process for a group of oxidized peptide fragments that can be used as “diagnostic markers” shown in FIG. 1, the “isoelectric focusing protein separation chip” is used when performing the “isoelectric focusing” 102 step. An "isoelectric point protein separation chip" composed of an electrophoresis apparatus that is compatible with isoelectric focusing and a drying apparatus that is compatible with lyophilization processing while being held in an "isoelectric point protein separation chip" FIG. 2 is a diagram schematically showing an overall configuration of an electrophoresis apparatus / drying apparatus conforming to FIG. FIG. 3 is a diagram illustrating a specific example of an “isoelectric point protein separation chip” schematically showing the configuration in FIG. 3, and shows an example of the configuration of an “isoelectric point protein separation chip” having a plurality of channels. FIG. FIG. 4 is a diagram illustrating a specific example of an electrophoresis apparatus / drying apparatus suitable for an “isoelectric point protein separation chip” schematically showing the entire configuration in FIG. 4, and is an image showing an appearance of the apparatus. In the step configuration of the search process for a group of oxidized peptide fragments that can be used as “diagnostic markers” shown in FIG. 1, fractionation was performed by “isoelectric focusing” in the step of “MALDI-TOF MS mass spectrometry” 103. The peak position (m / z) _peak of the ionic species [M _peptide + H] ⁺ derived from a plurality of types of peptide fragments (pI, M _peptide ) contained in the “single compartment (pI)” is measured. It is a figure which shows typically operation which specifies based on the "MALDI-TOF MS mass spectrometry" spectrum, and operation which _calculates | requires the peak intensity I (pI, [M _peptide + H] ^< +>). In the step configuration of the search process for a group of oxidized peptide fragments that can be used as “diagnostic markers” shown in FIG. 1, each peptide fragment in the “isoelectric focusing” step 102 in the “sample profiling” step 104 (PI, M _peptide ) spot "spreading direction of flow path" is the signal intensity I of the "MALDI-TOF MS mass spectrometry" spectrum measured in a plurality of consecutive "single sections (pI)". (pI, m / z) a, (pI, m / z) is plotted on the surface, each peptide fragment (pI, M _peptide) to determine the pI _mean of the spot center of each peptide fragment (pI, M _peptide) ionic species derived from the [M _peptide + H] specifying the _{^{+ (pI mean, (m /}} z) peak) and, the ionic species [M _peptide ⁺ H] + of total peak intensity I (pI, [M _peptide + ] ⁺⁾ Is a diagram schematically showing an operation for obtaining the _total. In Table 2, tryptic enzyme digestion of ApoB100 protein, which gives an ionic species [M _peptide + H] ⁺ of m / z = 1306, described as a peptide fragment that can be used as “diagnostic marker that can be used for diagnosis of heart disease” For the peptide fragment (amino acid number 4487-4496 fragment) of the amino acid sequence IISDYHQQFR that does not contain the Lys residue and the Met residue, 10 patients who were diagnosed with ischemic heart disease (number of coronary artery lesion branches: 3), In a sample derived from 23 non-patient groups (0 coronary artery lesion branch number), a total of 33 patients, the normalized peak intensity of the ion species [M _peptide + H] ⁺ of m / z = 1306 was increased to a total of 33 persons. In the derived sample group, the Z-scored value (Z score) is a sample derived from 10 patients group (coronary artery lesion branch number 3), non-patient group (coronary artery lesion branch number 0) and 23 sun samples. FIG. 9 is a graph in which the two subgroups of the pull are divided and plotted, and shows that there is a statistically significant difference in the distribution of the Z score values (Z scores) between the two subgroups. is there. Modification to the amino group (—NH ₂ ) of the Lys side chain by malondialdehyde, specifically, MDA-modified Lys generated by MDA conversion of the amino group (—NH ₂ ), which is a modification involving one MDA molecule , and corresponds to a modification MDA bimolecular is involved is a diagram showing the molecular structure of DHP of Lys produced by DHP (dihydroxypyridine) of an amino group (-NH _2). It is a figure which shows the 1-1000th range in the amino acid sequence of ApoB100 protein. It is a figure which shows the 1001-2000th range in the amino acid sequence of ApoB100 protein. It is a figure which shows the 2001-3000th range in the amino acid sequence of ApoB100 protein. It is a figure which shows the 3001-4000th range in the amino acid sequence of ApoB100 protein. It is a figure which shows the 4001-4563rd range in the amino acid sequence of ApoB100 protein.

  Below, it demonstrates in detail regarding the 1st form-3rd form of this invention.

  First, in all of the first to third aspects of the present invention, the present inventors have developed the progression of atherosclerosis in the coronary arteries, which is the main cause of heart disease, particularly ischemic heart disease. This corresponds to an invention that uses a newly found “cardiac disease diagnostic marker” as one of the indexes reflecting the disease state when evaluating the degree.

  The “cardiac disease diagnostic marker” used in the present invention shows a clear “quantitative change” reflecting the progression of the pathological condition of ischemic heart disease in a patient (subject) by the following search procedure. It has been identified as a “marker”. Specifically, a group of patients (subjects) with a high degree of progression of the pathological condition of ischemic heart disease, specifically, a group of patients determined to have 3 coronary lesions, and a healthy group determined to have 0 coronary lesions When comparing the normal (non-patient) group, the amount detected in the patient group is “significantly high” or “significantly low” based on the amount detected in the healthy (non-patient) group These are specified as “markers” indicating statistical test results.

  The “cardiac disease diagnostic marker” used in the present invention, its search procedure, and the identified “cardiac disease diagnostic marker” are actually based on the amount detected in the healthy (non-patient) group. The amount detected in the patient group will be described in more detail for the results of statistical tests that are “significantly high” or “significantly low”.

As an index reflecting the degree of progression of the pathological condition of ischemic heart disease, the present inventors have _compared the ratio of the _non- oxidized LDL content concentration C _LDL and the oxidized LDL content concentration C _OxLDL in the blood collected from the subject. , (C _OxLDL / C _LDL ) is focused on the fact that it has already been verified that it can actually be used (see Non-Patent Document 1). In addition, attention was also paid to the fact that oxidative LDL, in particular, MDA-LDL, has been reported to verify that it is involved in the early lesions of arteriosclerosis (see Non-Patent Document 2). Therefore, the concentration of MDA-LDL in the blood collected from the subject as an index reflecting the degree of progression of ischemic heart disease, specifically, the degree of progression of atherosclerosis in the coronary artery C _MDA− The ratio of the content concentration C _LDL of LDL to non-oxidized _LDL was also judged to be usable.

At that time, in the process of producing MDA-LDL from LDL in the body, ApoB100 protein containing MDA contained in LDL, which is produced along with oxidation of LDL, in particular, the Lys residue in the ApoB100 protein And the modification to the amino group (—NH ₂ ) of the Lys residue side chain occurs. Specifically, as shown in FIG. 10, the Lys residue is a modification involving one MDA molecule, which is an MDA-modified Lys residue generated by MDA conversion of an amino group (—NH ₂ ), or a plurality of MDA residues. It is converted to a DHP-modified Lys residue generated by DHP (dihydroxypyridine) conversion of an amino group (—NH ₂ ) corresponding to a modification involving a molecule.

Since oxidation of LDL progresses due to “oxidative stress”, oxidation of amino acid residues in the ApoB100 protein contained in LDL, for example, —CH ₂ —S—CH ₃ of the Met residue side chain also progresses, It is converted into —CH ₂ —SO—CH ₃ (monoxide: Met (O)) and —CH ₂ —SO ₂ —CH ₃ (dioxide: Met (O ₂ )).

  The higher the level of “oxidative stress”, the more LDA oxidation proceeds, so the amount of MDA-LDL produced increases. At the same time, the amount of modification by MDA in the ApoB100 protein contained in the produced MDA-LDL, specifically In addition, the content ratio of MDA-modified Lys residues and DHP-modified Lys residues also increases. Of course, the higher the level of “oxidative stress”, the higher the content ratio of Met residues subjected to oxidative modification in the ApoB100 protein contained in the generated MDA-LDL.

  The present inventors fractionated MDA-LDL contained in blood collected from a subject, and modified amount by MDA in ApoB100 protein contained in the fractionated MDA-LDL, specifically, Based on the measurement results of the content ratio of the MDA-modified Lys residue and the DHP-modified Lys residue, in the process of generating MDA-LDL from LDL, the level of “oxidative stress” was high, or the level of “oxidative stress” was It came to the idea that it was possible to determine whether the environment was low. In this method, it is not necessary to collect the entire amount of MDA-LDL contained in the blood collected from the subject. A part of the MDA-LDL is sampled, and the MDA-LDL contained in this sample is subjected to MDA-modified Lys. A form in which the content ratio of residues, DHP-modified Lys residues, or the content ratio of Met residues subjected to oxidative modification can be used.

  The ApoB100 protein contained in LDL forms a tertiary structure, and among the plurality of Lys residues contained in the ApoB100 protein, those located inside the tertiary structure are not modified by MDA, and are tertiary. Lys residues exposed on the surface of the structure are modified by MDA. In addition, among the plurality of Met residues contained in the ApoB100 protein, those located inside the tertiary structure are not subjected to oxidative modification, and Met residues exposed on the surface of the tertiary structure are subject to oxidative modification. receive.

  Furthermore, even among the plurality of Lys residues exposed on the tertiary structure surface of the ApoB100 protein, there is a difference in the probability of modification by MDA, so depending on the level of “oxidative stress” In the entire ApoB100 protein, the content ratio of MDA-modified Lys residues and DHP-modified Lys residues changes. Further, among the plurality of Met residues exposed on the tertiary structure surface of the ApoB100 protein, there is a difference in the probability of undergoing oxidative modification. Therefore, depending on the level of “oxidative stress”, ApoB100 In the whole protein, the content ratio of Met residues subjected to oxidative modification changes.

  In other words, instead of the measurement result of the content ratio of the MDA-modified Lys residue and the DHP-modified Lys residue in the entire ApoB100 protein contained in MDA-LDL, a plurality of the plurality of ApoB100 protein exposed on the tertiary structure surface Among the Lys residues, a specific Lys residue site whose probability of modification by MDA varies clearly depending on the level of “oxidative stress” is selected, and the selected Lys residue site Determine the level of “oxidative stress” in the process of producing MDA-LDL from LDL based on the measurement result of the ratio of MDA-Lys residue converted to MDA-modified Lys residue I realized that this is possible. Moreover, instead of the measurement result of the content ratio of Met residues subjected to oxidative modification in the entire ApoB100 protein contained in MDA-LDL, a plurality of Met residues exposed on the tertiary structure surface of ApoB100 protein Of these, depending on the level of “oxidative stress”, a specific Met residue site that clearly varies the probability of undergoing oxidative modification is selected, and the oxidative modification at the selected Met residue site is selected. Based on the measurement result of the ratio of conversion to Met residues received, it was conceived that the level of “oxidative stress” in the process of generating MDA-LDL from LDL can be determined.

  In addition, when the content ratio of the MDA-modified Lys residue and the DHP-modified Lys residue is increased in the entire ApoB100 protein, the Lys residue at the cleavage site, which is inherently susceptible to trypsin digestion, becomes MDA-modified Lys residue, DHP-modified Lys residue Probability of being converted to the base increases. At that time, the probability of trypsin digestion at sites that are inherently susceptible to trypsin digestion is reduced due to conversion to MDA-modified Lys residues and DHP-modified Lys residues. On the other hand, it has been conceived that the probability of trypsin digestion at Lys and Arg residues present around such sites may be relatively increased. In other words, depending on the level of “oxidative stress”, the probability that the Lys residue at the cleavage site, which is inherently susceptible to trypsin digestion, is subject to oxidative modification varies, resulting in Lys existing around the site. Variation of trypsin digestion efficiency at the residue, Arg residue may be induced. Therefore, based on the measurement result of the relative change in the amount of peptide fragments whose production efficiency increases with the relative increase in trypsin digestion efficiency, which is induced by the increase in the level of “oxidative stress”, LDL to MDA − It has been thought that it is possible to determine the level of “oxidative stress” in the process of generating LDL. It should be noted that the peptide fragment itself, whose generation efficiency increases with the relative increase in trypsin digestion efficiency, is an unmodified Lys residue, Arg residue, and further in the peptide fragment It has also been conceived that there is no need for MDA-modified Lys residue, DHP-modified Lys residue, or Met residue subjected to oxidative modification to be inherent in the Met residue.

  For example, the level of “oxidative stress” even in the case of a peptide fragment that does not contain “MDA-modified Lys residue, DHP-modified Lys residue, and Met residue subjected to oxidative modification” described in Table 13 to be described later When the fluctuation of trypsin digestion efficiency in the Lys residue and Arg residue existing around the peptide fragment is induced, the generation efficiency of such peptide fragment may be relatively increased. I also thought.

  The “cardiac disease diagnostic marker” used in the present invention is based on the selection criteria described above, depending on the level of “oxidative stress”, the probability of being modified by MDA at the amino acid residue site, or , Corresponding to a group of peptide fragments derived from ApoB100 protein contained in MDA-LDL, including specific amino acid residue sites selected as sites that clearly change the probability of undergoing oxidative modification.

  MDA at the amino acid residue site in the peptide fragment derived from ApoB100 protein contained in MDA-LDL, which is the “cardiac disease diagnostic marker” used in the present invention and contains the above-mentioned specific amino acid residue site The measurement result of the probability of undergoing modification by or the probability of undergoing oxidative modification, of course, reflects the progress of oxidation in the ApoB100 protein contained in the fractionated MDA-LDL.

  Therefore, the first aspect of the present invention is the probability that a peptide fragment containing a specific amino acid residue site selected as a “cardiac disease diagnostic marker” has been modified by MDA at the amino acid residue site. Alternatively, based on the measurement result of the probability of undergoing oxidative modification, oxidation of amino acid residues in ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject, and by MDA It is an invention of a method for evaluating the degree of progress of modification (the degree of progress of oxidation).

  The second aspect of the present invention is the probability that a peptide fragment containing a specific amino acid residue site selected as a “cardiac disease diagnostic marker” has been modified by MDA at the amino acid residue site, or Based on the measurement result of the probability of undergoing oxidative modification, the “oxidation progress” is evaluated, and the MDA-LDL to be fractionated is generated from the LDL using the evaluation result of the “oxidation progress”. Is an invention of a method for estimating the level of “oxidative stress level” in the process. That is, the method for estimating the level of the “oxidative stress level” according to the second aspect of the present invention is a method for estimating the level of the “oxidative stress level” in the body of the subject who has collected the blood sample to be evaluated. It corresponds.

  As a third aspect of the present invention, when evaluating the degree of progression of atherosclerosis in the coronary artery, which is a major factor of heart disease, particularly ischemic heart disease, The invention is intended to use the estimation result of the level of “oxidative stress level” in the body of the subject. At that time, in the third aspect of the present invention, a method for estimating the level of “oxidation stress level” according to the second aspect of the present invention, that is, “inside the body of the subject from whom the blood sample to be evaluated was collected” A method for estimating the level of the “oxidative stress level” is applied to estimate the level of the “oxidative stress level” in the body of the subject. In the second embodiment of the present invention, the progress of oxidation in the ApoB100 protein contained in the MDA-LDL collected from the blood sample collected from the subject is evaluated, and the “progress of oxidation” is evaluated. Based on the evaluation results, the level of “oxidation stress level” is estimated. In the method of evaluating the “degree of oxidation progress” according to the second aspect of the present invention, the selected peptide fragment containing a specific amino acid residue site is selected based on the above selection criteria. It is used as a “marker” suitable for evaluation.

  The third aspect of the present invention is an MDA fractionated from a blood sample collected from a subject, which is carried out for the purpose of grasping the pathological condition of the subject prior to evaluation of the degree of progression of atherosclerosis in the coronary artery. -A peptide containing a specific amino acid residue site selected based on the above-mentioned selection criteria for "markers" used for the evaluation of the progress of oxidation in ApoB100 protein contained in LDL It is an invention of a method of selecting from a group of fragments. Therefore, the third aspect of the present invention corresponds to “a method for selecting a diagnostic marker that can be used for heart disease diagnosis” from a group of “cardiac disease diagnosis markers” selected based on the above-mentioned selection criteria. Yes.

  Prior to evaluation of the degree of progression of atherosclerosis in the coronary artery, ApoB100 protein contained in MDA-LDL fractionated from a blood sample collected from a subject, which is carried out for the purpose of grasping the pathology of the subject The degree of oxidation progress is evaluated. At that time, as described above, as a “marker” used for the evaluation, the content ratio of MDA-modified Lys residue, DHP-modified Lys residue, and oxidative modification in the entire ApoB100 protein contained in MDA-LDL Any of the “markers” that can estimate the content ratio of received Met residues or the content ratio of other amino acid residues (for example, Tyr residues) subjected to oxidative modification with high accuracy can be used. is there. Among the various groups of “markers”, in the third embodiment of the present invention, based on the above selection criteria, “markers” used for evaluation are selected from a group of selected “cardiac disease diagnostic markers”. There is a technical feature in this respect.

  Based on the above selection criteria, the procedure for selecting “cardiac disease diagnostic markers” used in the present invention will be described below.

  FIG. 1 shows the overall procedure of the “cardiac disease diagnostic marker” selection procedure, that is, the “marker search” process. The process includes “sample pretreatment” step 101, “isoelectric focusing” step 102, “MALDI-TOF MS mass spectrometry” step 103, “sample profiling” step 104, and “data analysis / marker identification” step 105. It consists of five steps.

  The final object of the present invention is to measure the amount of the selected “cardiac disease diagnosis marker” using a blood sample collected from a subject prior to “cardiac disease diagnosis”. Therefore, the conditions of “sample pretreatment”, “isoelectric focusing”, and “MALDI-TOF MS mass spectrometry” are suitable for measurement using a small amount of blood sample in consideration of the amount of blood sample collected from the subject. Under the conditions, “cardiac disease diagnostic marker” is selected. Therefore, the same conditions as those used for “sample pretreatment”, “isoelectric focusing”, and “MALDI-TOF MS mass spectrometry” employed in the selection of “cardiac disease diagnostic markers” are used. Prior to “diagnosis”, a blood sample collected from a subject can be used to measure the amount.

  Specifically, in the “isoelectric focusing” step, “isoelectric focusing” using an “isoelectric focusing protein separation chip” described later is performed, and then the “isoelectric focusing protein separation chip”. In this state, a drying process is performed in a state of being held in a “insulator”, and “MALDI-TOF MS mass spectrometry” is performed in a state of being held in a “isoelectric point protein separation chip” that has been subjected to a drying process.

(1) “Sample pretreatment” step “Sample pretreatment” step 101 has four steps as shown in FIG. 2: “serum preparation” step 201 for preparing “serum” from a blood sample; anti-MDA-LDL antibody is used. “Immunoprecipitation” step 202 for fractionating MDA-LDL from serum using the “immunoprecipitation” method; ApoB100 protein contained in the fractionated MDA-LDL using a trypsin digestion treatment "Fragmentation" processing step 203 for preparing peptide fragments derived from the solution; removing the solvent in the trypsin digestion reaction solution containing the prepared peptide fragments, and collecting them as a dry product containing the total amount of the prepared peptide fragments The “fragmented peptide recovery” step 204 is performed.

(1-1) "Serum preparation" step 201
First, a subject for selecting “cardiac disease diagnostic marker” is ApoB100 protein contained in MDA-LDL, which is contained in blood collected from a subject. Since MDA-LDL is dissolved in blood serum, serum is prepared from a blood sample collected from a subject according to a standard method. After the blood coagulation treatment, the clot (precipitate) is separated by centrifugation and the serum is collected.

  When the collected serum is stored, it is stored frozen at a temperature of −80 ° C. or lower. That is, by performing the cryopreservation, MDA and oxidation are further prevented from proceeding with respect to MDA-LDL contained in the serum during the preservation.

(1-2) "Immunoprecipitation" step 202
Various soluble proteins and lipids are dissolved in the collected serum. In particular, in addition to MDA-LDL, oxidized LDL that has not been converted to MDA and LDL that has not undergone oxidation also coexist in the serum. In order to selectively collect only MDA-LDL from serum, an “immunoprecipitation” method using an anti-MDA-LDL antibody is used.

  An affinity carrier having an anti-MDA-LDL antibody immobilized on a carrier is added to the serum, and only MDA-LDL is selectively reacted with the anti-MDA-LDL antibody by an antigen-antibody reaction and captured. Thereafter, by collecting the affinity carrier by solid-liquid separation, only MDA-LDL contained in the serum is fractionated.

  In addition, although the density | concentration of MDA-LDL contained in serum is unknown, since it is not necessary to collect | recover the whole quantity of MDA-LDL contained in serum, the quantity of the affinity carrier to be used normally Can be selected such that the amount of anti-MDA-LDL antibody added per 100 μL of serum is in the range of 2 μg to 4 μg. As a carrier for immobilizing the anti-MDA-LDL antibody, for example, when magnetic beads suitable for immobilization of IgG antibody are used, when the affinity carrier is recovered by solid-liquid separation after the antigen-antibody reaction, Is preferable because it can be selectively recovered.

  In serum, in addition to MDA-LDL, LDL that has not undergone MDA conversion, for example, LDL that has not undergone any modification due to oxidation coexists in large quantities. In general, the concentration of oxidized LDL contained in serum is 10 μg / dL or more and less than 100 μg / dL, but the concentration of all LDL is reported to be about 50 to 100 mg / dL. Therefore, the ratio of the MDA-LDL content concentration C [MDA-LDL] and the total LDL content concentration C [totalLDL] in the serum; C [MDA-LDL] / C [totalLDL] is usually C [MDA -LDL] / C [totalLDL] <0.2%. When recovering the affinity carrier, non-selective adsorption of LDL that has not undergone MDA conversion, for example, LDL that has not undergone any modification due to oxidation, is prevented. Thereafter, the affinity carrier is thoroughly washed. When sufficient washing is completed, only the unreacted anti-MDA-LDL antibody and the anti-MDA-LDL antibody that forms a complex with MDA-LDL are immobilized on the surface of the recovered affinity carrier. ing.

  As the anti-MDA-LDL antibody used in the “immunoprecipitation treatment” step 202, for example, an anti-MDA-LDL monoclonal antibody such as antibody No. 29225 (Sekisui Medical) is suitable.

  A method for creating an anti-human MDA-LDL monoclonal antibody by immunizing a mouse using human MDA-LDL as an immunogen has already been reported (see Japanese Patent No. 315587). Among the anti-human MDA-LDL monoclonal antibodies created by this technique, those showing specific reactivity with human ApoB100 protein contained in human MDA-LDL are selected, and the “immunoprecipitation treatment” The anti-MDA-LDL antibody used in step 202 can be used.

(1-3) “fragmentation” processing step 203
After washing, MDA-LDL immobilized on the surface of the recovered affinity carrier and forming a complex with the anti-MDA-LDL antibody was enzymatically digested with trypsin, and separated MDA-LDL A group of peptide fragments derived from the ApoB100 protein contained therein is prepared.

  Prior to enzymatic digestion, MDA-LDL immobilized on the surface of the affinity carrier, forming a complex with the anti-MDA-LDL antibody, was separated from the antibody and immobilized on the surface of the affinity carrier. It is generally desirable to subject the anti-MDA-LDL antibody to solid-liquid separation and to recover MDA-LDL in the liquid phase.

  As a means for releasing the antigen from the antibody, a treatment for reducing the reactivity of the antibody to the antigen is used. Usually, the binding between an epitope (antigenic determinant) present in an antigen and a CDR (complementarity determining region) in an antibody is due to a hydrophobic interaction. For example, in order to reduce the interaction between the epitope (antigenic determinant) present in the antigen and the CDR (complementarity determining region) in the antibody, a polar organic solvent such as acetonitrile is added to the CDR ( Means for solvating (coating) the complementarity determining region) with the polar organic solvent can be used. In addition, by adding a surfactant-type protein denaturing agent such as SDS, it is possible to use a method of denaturing the variable region of the antibody and eliminating the affinity for the epitope (antigenic determinant) present in the antigen. is there.

  On the other hand, without separating MDA-LDL from the antibody, ApoB100 protein contained in MDA-LDL is made complex with anti-MDA-LDL antibody by trypsin acting on MDA-LDL. It is also possible to adopt a form in which a group of derived peptide fragments is prepared. In that case, in addition to the peptide fragment group derived from ApoB100 protein contained in MDA-LDL, the peptide fragment group derived from an anti-MDA-LDL antibody is also generated. Since the peptide fragment group derived from the anti-MDA-LDL antibody can be specified separately, from the mixture of the peptide fragment group derived from the ApoB100 protein contained in the MDA-LDL and the peptide fragment group derived from the anti-MDA-LDL antibody, MDA-LDL It is possible to specify a peptide fragment group derived from the ApoB100 protein contained therein.

  In addition, when performing enzymatic digestion using trypsin, trypsin-derived peptide fragment groups are also generated due to trypsin autolysis. Since a trypsin-derived peptide fragment group resulting from this self-digestion of trypsin can also be specified separately, a peptide fragment group derived from the ApoB100 protein contained in MDA-LDL, a peptide fragment group derived from an anti-MDA-LDL antibody, derived from trypsin It is possible to specify the peptide fragment group derived from ApoB100 protein contained in MDA-LDL from the mixture of the peptide fragment groups.

  In the “fragmentation” processing step 203, the entire amount of ApoB100 protein contained in the fractionated MDA-LDL is fragmented by enzymatic digestion using trypsin. At that time, trypsin cleaves the peptide bond at the C-terminal side of the Arg residue and the peptide bond at the C-terminal side of the Lys residue. Generally, trypsin cleaves the MDA-modified Lys residue and the DHP-modified Lys residue at the C-terminal side. The efficiency at which the peptide bond is cleaved is lower than the efficiency at which the peptide bond at the C-terminal side of the unmodified Lys residue is cleaved. When examining the ratio of Lys residues at specific sites converted to MDA-modified Lys residues and DHP-modified Lys residues, it is desirable to select reaction conditions that make the difference in the cleavage efficiency clear. . That is, cleavage of the peptide bond on the C-terminal side of the Arg residue, and the cleavage of the peptide bond on the C-terminal side of the unmodified Lys residue are completed, but the C-terminal side of the MDA-modified Lys residue and the DHP-modified Lys residue It is desirable to select reaction conditions in which the progress rate of cleavage of the peptide bond is clearly inferior.

  Therefore, the reaction conditions for enzyme digestion using trypsin are selected such that the trypsin concentration in the reaction solution is in the range of 50 ng / μL to 100 ng / μL, and the reaction temperature is stirred and the liquid temperature is 35 ° C. to 38 ° C. It is desirable to select the reaction time in the range of 12 hours to 24 hours. In addition, for example, when performing enzyme digestion using trypsin on ApoB100 protein contained in MDA-LDL fractionated from 300 μL of serum, it is desirable to select the total amount of the reaction solution in the range of 15 μL to 40 μL. .

  When the treatment for separating MDA-LDL from the antibody is performed, the enzyme digestion reaction solution using trypsin contains a peptide fragment group derived from ApoB100 protein, a peptide fragment group derived from trypsin, and trypsin contained in MDA-LDL. It is out.

  When using MDA-LDL that forms a complex with an anti-MDA-LDL antibody without separating MDA-LDL from the antibody, an enzyme digestion reaction solution using trypsin is included in MDA-LDL A peptide fragment group derived from ApoB100 protein, a peptide fragment group derived from anti-MDA-LDL antibody, a peptide fragment group derived from trypsin, and an affinity carrier carrying trypsin and anti-MDA-LDL antibody. When the affinity carrier carrying the anti-MDA-LDL antibody is removed by solid-liquid separation, the remaining liquid phase contains a group of peptide fragments derived from the ApoB100 protein contained in the MDA-LDL, derived from the anti-MDA-LDL antibody. A peptide fragment group, a trypsin-derived peptide fragment group, and trypsin are included.

  In either case, the liquid phase portion containing the entire amount of the peptide fragment group derived from the anti-MDA-LDL antibody is recovered as a sample solution that has been subjected to the “fragmentation” treatment. The amount of the “fragmented” treated sample solution to be collected is substantially equal to the amount of the entire enzyme digestion reaction solution.

(1-4) “Fragmented peptide recovery” step 204
The solvent contained in the collected “fragmented” treated sample solution is removed, and collected as a dried product containing the total amount of the prepared peptide fragments.

  When the amount of the collected “fragmented” sample solution is in the range of 15 μL to 40 μL, for example, a centrifugal evaporator can be used for drying by removing the solvent. When the dried product containing the total amount of the prepared peptide fragment group is stored after the drying treatment, for example, it is stored refrigerated at a temperature of −30 ° C. or lower.

(2) “Isoelectric focusing” step Generally, since the concentration of oxidized LDL contained in serum is 10 μg / dL or more and less than 100 μg / dL, oxidized LDL contained in 300 μL of serum is 30 ng or more and less than 300 ng. The weight ratio of the ApoB100 protein contained in the oxidized LDL1 particles is about 25% by mass, and when the contained oxidized LDL is 30 ng or more and less than 300 ng, the contained ApoB100 protein is 8 ng or more and less than 80 ng. Estimated.

In the collected “dried product containing the total amount of prepared peptide fragments” sample, ApoB100 protein contained in MDA-LDL in the range of 8 ng or more and less than 80 ng in total per sample (per 300 μL of serum) The peptide fragment group derived from is contained. In other words, a group of peptide fragments obtained by trypsin digesting ApoB100 protein contained in MDA-LDL in a range of 0.015 pmol or more and less than 0.15 pmol per sample (per 300 μL of serum) is contained. . Each peptide fragment derived from ApoB100 protein contained in one sample and contained in MDA-LDL is cut out from one molecule of ApoB100 protein. Therefore, the number of molecules of each peptide fragment is the number of molecules of ApoB100 protein. Number or less. Therefore, the content M (pI, M _peptide ) of each peptide fragment (pI, M _peptide ) contained in one sample prepared from 300 μL of serum is in the range of 0.015 pmol or more and less than 0.15 pmol. It is estimated that

On the other hand, are separated by "isoelectric focusing", the spots of each peptide fragment (pI, M _peptide), the peptide fragment (pI, M _peptide) ionic species derived from the [M _peptide ⁺ H] + of the "m The distribution of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ , indicating the situation of “/ z” value and “spreading in the flow path direction” at each spot, It is necessary to measure with sufficient accuracy and accuracy using "-MS mass spectrometry". At that time, contained in one sample, the total amount M of each peptide fragment _{(pI, M peptide) (pI} , M peptide) is spread sigma _pI of spots "flow path direction of each peptide fragment (pI, M _peptide) ”And the product of the width W of the flow path, it can be considered to exist within the spot area S (pI, M _peptide ) indicated by (W × σ _pI ). M (pI, M _peptide ) / S (pI, M _peptide ) corresponds to the average areal density of peptide fragments (pI, M _peptide ) in the spot. The distribution of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ion species [M _peptide + H] ⁺ , which indicates the situation of “spreading in the direction of the flow path” at each spot, In order to use and measure with sufficient accuracy and accuracy, the average surface density of peptide fragments (pI, M _peptide ) at the spot, M (pI, M _peptide ) / S (pI, M _peptide ) It is necessary to be at a sufficient level.

Therefore, when performing the "isoelectric focusing", by using the "isoelectric point protein separation chip", by narrowing the spot area S (pI, M _peptide) of the peptide fragment (pI, M _peptide) The average areal density of peptide fragments (pI, M _peptide ) and M (pI, M _peptide ) / S (pI, M _peptide ) are set to a sufficient level. Specifically, by setting the length L of the channel of the “isoelectric point protein separation chip” to 35 mm and separating the peptide fragments having a pI in the range of 3 to 10 along the channel into respective spots. When the “span in the flow path direction σ _pI ” is about σ _pI = 0.2, the length δL of the spot is about 1 mm.

  In the “isoelectric focusing” step 102, a peptide fragment group derived from the ApoB100 protein contained in MDA-LDL, which is contained in the collected “dried product containing the total amount of the prepared peptide fragments” sample, anti-MDA -The peptide fragment group derived from LDL antibody, the peptide fragment group derived from trypsin, and trypsin are separated into respective spots by isoelectric focusing. Thereafter, the entire “isoelectric point protein separation chip” is cooled to −30 ° C. while keeping the separated spots along the flow path of the “isoelectric point protein separation chip”. Freeze the “sample solution after electrophoresis”. Furthermore, freeze-drying is performed while the frozen “sample solution after electrophoresis” is held in the flow path. As a result of the lyophilization treatment, the spot separated along the flow path of the “isoelectric point protein separation chip” is converted into a lyophilized peptide fragment spot at that position.

The “spot of peptide fragments that have been lyophilized” indicates “spreading direction σ _{pI in the} flow path direction” centering on the isoelectric point pI of the peptide fragment (pI, M _peptide ). That is, the areal density of the lyophilized peptide fragments constituting the spot shows a change in the flow direction that can be approximated to a Gaussian distribution centered on the isoelectric point pI, and the standard deviation of the Gaussian distribution is “ This corresponds to the spread σ _{pI in the} flow path direction.

By applying “MALDI-MS mass spectrometry”, the ionic strength I (pI, [M _peptide + H] of the ionic species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) forming a spot is formed. ] ⁺ ) And analyzing the distribution in the channel direction, the center pI _mean of the spot can be specified. Further, assuming a Gaussian distribution centered on the pI _mean , a standard deviation capable of approximating the measured ionic strength I (pI, [M _peptide + H] ⁺ ) distribution is calculated, and “a spread in the channel direction σ _pI Is possible.

In “isoelectric focusing”, peptide fragments having a pI in the range of 3 to 10 are separated into respective spots along the flow path. The interval is divided into 71 sections, and the m / z of ionic species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) present in each section, and its ionic strength I (pI , [M _peptide + H] ⁺ ). The difference ΔpI of pI of each section can be considered as (10−3) /70=0.1. Actually, when “isoelectric focusing” is performed, the pH gradient (dpH / dL) formed along the flow path shows a slight variation, so that pI is included in the sample solution for electrophoresis. A plurality of known peptides and proteins are added as pI standard substances, and the pI of each spot position is estimated along the flow path with reference to the spot positions of the plural kinds of pI standard substances.

On the other hand, “MALDI-MS mass spectrometry” is applied to measure the ion species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) present in each compartment. The length ΔL of each section is set so as to satisfy ΔL ≧ d _laser with reference to the irradiation spot diameter d _laser of the laser beam used by the “MALDI-MS mass spectrometer”. At the same time, the width W of the flow path is also set so as to satisfy W ≧ d _{laser based} on the irradiation spot diameter d _laser of the laser light. The measurement of the ion species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) present in each section is performed by irradiating the center of each section with a laser beam having an irradiation spot diameter d _laser. Irradiation spot diameter d Peptide fragments (pI, M _peptide ) present in the _laser are ionized.

  The configuration of the “isoelectric focusing protein separation chip” and “electrophoresis / drying device” used in the “isoelectric focusing” step 102 and the outline of the usage thereof will be described below.

(2-1) "Isoelectric point protein separation chip"
FIG. 3 schematically shows an example of the configuration of an “isoelectric point protein separation chip” used in the “isoelectric focusing” step 102. The configuration example schematically shown in FIG. 3 is a single channel configuration in which one channel 302 is provided. However, in order to perform “isoelectric focusing” on a plurality of samples simultaneously, A multi-channel configuration in which the channels 302 are arranged in parallel can also be adopted. 3A is a top view of the lid 304, FIG. 3B is a top view of the substrate 303, and FIG. 3C is a cross-sectional view of the assembled chip 300 along AA ′. Each is shown. FIG. 3D shows an enlarged view of the flow path 302 formed on the substrate 303. A plurality of hydrophilic columnar bodies 301 are formed on the bottom surface portion of the channel 302, and side wall portions are formed on both sides of the bottom surface portion to constitute a groove-like structure. Reservoirs 305 a and 305 b are provided at both ends of the flow path 302.

  The lid 304 includes a flat lid base portion 306 and a lid resin portion 307 that is located on the lower surface of the lid base portion 306 and contacts the substrate 303. The lid 304 has through holes for solution injection at positions corresponding to the reservoirs 305a and 305b at both ends of the flow path 302. In the assembled chip 300, when the lid 304 is overlaid on the upper surface of the substrate 303, the reservoirs 305a and 305b at both ends of the flow path 302 are aligned with the through holes for solution injection formed in the lid 304. At that time, the upper surface of the flow path 302 is covered with the lid resin portion 307 of the lid 304. The surface of the lid 304 that covers the upper surface of the channel 302 and is in contact with at least the sample solution introduced into the channel 302 is hydrophobic or water-repellent. In particular, the entire surface of the lower surface of the lid resin portion 307 of the lid 304 is preferably made hydrophobic or water repellent.

  When “isoelectric focusing” is performed, a high voltage is applied to the sample solution introduced into the flow path 302 using both ends of the flow path 302. A potential gradient (electric field) along the flow path 302 is formed in the sample solution introduced into the flow path 302 by the applied high voltage. Therefore, it is necessary to insulate the substrate 303 from the sample solution introduced into the flow path 302. Therefore, it is desirable to manufacture the substrate 303 itself using a highly insulating material. Further, since the lower surface of the lid resin portion 307 of the lid 304 is also in contact with the sample solution introduced into the channel 302, the lid resin portion 307 of the lid 304 is insulated from the sample solution introduced into the channel 302. It is necessary to keep. Therefore, it is desirable to produce at least the lid resin portion 307 of the lid 304 using a highly insulating material. In addition, it is desirable that the base portion 306 itself of the lid 304 is also made using a highly insulating material.

  Since the flow path 302 is formed on the substrate 303, it is desirable to use a highly insulating material that can be processed with sufficient accuracy when the flow path structure is processed. As an example of a highly insulating material that satisfies the above requirements, quartz or glass, and plastic materials having high insulating properties such as PC (polycarbonate) and PMMA (polymethyl methacrylate) can be given.

  Note that the flow path 302 illustrated in FIG. 3 is a straight flow path, and has a shape suitable for forming a potential gradient (electric field) along the flow path 302 in the sample solution introduced into the flow path 302. is there. However, it is possible to form a potential gradient (electric field) along the channel 302 in the sample solution introduced into the channel 302 by providing a plurality of channels on the upper surface of the substrate 303. As long as it is, a planar shape other than the linear flow path can be adopted. The planar shape of the flow path 302 itself, the arrangement of the flow paths, and the length of the flow paths are appropriately selected according to the sample used and the electrophoresis conditions. Specifically, the cross-sectional shape of the flow path 302 is preferably selected so that the width W of the flow path and the depth D of the flow path are 3 μm to 2000 μm. The total length L of the flow path, the width W of the flow path, and the depth D of the flow path are selected within a range that can satisfy the requirements for the amount of peptide fragments per unit bottom area required for performing mass spectrometry. . At the same time, the size of the flow path must be selected within the range that enables temperature control with high thermal efficiency and high-efficiency fluid control sufficient to dissipate Joule heat generated in the flow path during "isoelectric focusing". There is also. The size of the flow path is selected in consideration of the above two requirements and considering the processing accuracy of the material used for manufacturing the substrate 303.

  As a material for forming the lid base material portion 306, a material that can be processed such as maintaining the mechanical strength of the lid or manufacturing a through hole and having excellent insulating properties is preferably used. For the production of the lid base material portion 306, for example, a polymer resin material such as quartz or glass, an acrylic resin such as polycarbonate, PMMA, or a silicone resin such as PDMS can be used.

  On the other hand, the lid resin portion 307 covers the upper surface of the flow channel, and is brought into close contact with the upper surface of the side wall portion of the flow channel and the lower surface of the lid resin portion 307 so that the sample solution filled in the flow channel is leaked and oozed out. A configuration that does not occur is adopted. At the same time, when the lid is peeled, at least the lid resin portion 307 of the lid 304 is elastically deformed to provide a bending structure at the boundary of the peeling.

  For this reason, it is preferable to use an insulating material having high elastic deformability for the production of the lid resin portion 307. For example, polyolefins such as PDMS, PTFE (polytetrafluoroethylene), PP (polypropylene), PE (polyethylene), and polyvinyl chloride are used as the insulating material having high elastic deformation property used for the base material of the lid resin portion 307. Or polyester. In addition, it is preferable that at least a region corresponding to the upper surface of the flow path in the lower surface of the lid resin portion 307 is a surface exhibiting hydrophobicity or water repellency. Therefore, in order to make the lower surface of the lid resin portion 307 have a hydrophobic or water-repellent surface, in particular, a coating with a water-repellent material such as a silicone resin such as PDMS or a fluorine-based resin such as PTFE can be suitably used. . Alternatively, an insulating material having water repellency and high elastic deformability may be used as the base material of the lid resin portion 307.

  In addition, at least one end portion of the outer shape of the lid 304 is provided with a portion protruding from the outer shape of the substrate 303. The overhanging one end is used for applying an external force for removing the lid. If an external force is applied to one end of the lid 304 while fixing and pressing the substrate 303, the lid peeling proceeds. For example, when the rectangular substrate 303 and the lid 304 are selected, when the peeling direction of the lid is selected in the long side direction of the substrate 303, the outer shape of the lid 304 is the length of the long side direction. Make it longer than the side. When an external force is applied to a portion of the lid 304 that protrudes in the long side direction, an action point of the external force is set at that portion. Furthermore, after the peeling of the cover 304 is completed, when performing the operation of holding the cover 304, moving it from the upper surface of the substrate 303, and removing it, an area for supporting the end of the cover 304 can be set as the overhanging portion. It becomes possible. Further, when peeling / removing the lid 304, it is also possible to select a form using a protruding portion in the short side direction of the lid 304 provided along the long side of the substrate 303 as a portion to which an external force is applied.

  Note that the adhesion strength between the upper surface of the substrate 303 and the lid resin layer 307 is such that the solution filled in the channel is not leaked or exuded from the channel 302 formed on the upper surface of the substrate 303. Although it is sufficient for this, peeling is progressed on this contact surface by applying a predetermined external force. Further, in maintaining a close contact state between the upper surface of the substrate 303 and the lid resin layer 307, the adhesiveness of the lid resin layer 307 itself is lowered and compensated in a form depending on the load of an external force that closely contacts the two. Is also possible.

  As a method of performing the above-described lid peeling, for example, a method disclosed in International Publication No. 2006/085539 can be adopted.

(2-2) "Electrophoresis and drying equipment"
The configuration of the electrophoresis / drying apparatus used for the operation of “isoelectric focusing” using the above-mentioned “isoelectric focusing protein separation chip” and the subsequent “freeze-drying treatment” will be described below.

  FIG. 4 is a diagram schematically showing an overall configuration of an electrophoresis / drying apparatus 400 compatible with an “isoelectric point protein separation chip”. The electrophoresis / drying device 400 includes at least a mechanism 401 that accommodates the chip, a power source 402 and an electrode unit 403 for applying a voltage to a flow path in the chip during “isoelectric focusing”, And a mechanism 404 for controlling the temperature. In addition, after “isoelectric focusing”, the peptide fragments and proteins separated on the flow path and forming each spot are freeze-dried while holding the spots, and “freeze-drying treatment” is performed. A drying mechanism 405 is provided for immobilization as a “spot of a completed peptide fragment”.

  After isoelectric focusing of the sample solution in the flow path of the “isoelectric point protein separation chip” using the electrophoresis / drying device 400, the temperature control mechanism 404 is used to remove the sample solution in the flow path. Quick freeze. By rapidly freezing, the peptide fragments forming each spot and the separated state of the protein are fixed in the sample solution in the flow path. After the quick freezing is finished, the lid 304 is peeled off from the substrate 303 and removed while maintaining the cooling temperature. As a result, the “freezing sample solution” frozen body held in the flow path formed on the substrate 303 is exposed. The frozen body contains a solidified liquid component (water), and the solidified liquid component (water) is removed using a drying mechanism 405 while being kept frozen in the flow path, and freeze-dried. Apply. During this lyophilization process, the temperature control device 404 is used to remove the liquid component (water) that has been kept frozen and solidified, using a drying mechanism 405 that includes a sealed tank, a decompression mechanism, and the like. The As a result of the freeze-drying process, each spot that is separated along the flow path of the “isoelectric point protein separation chip” retains its position as a “spot of the peptide fragment that has been freeze-dried”. Fixed dry.

  Next, each mechanism and its function constituting an electrophoresis / drying apparatus suitable for an “isoelectric point protein separation chip” will be described with more specific examples. The configuration of the electrophoresis / drying apparatus suitable for the “isoelectric point protein separation chip” is not limited to the configuration shown in the following specific embodiment.

  When performing “isoelectric focusing”, the close contact state between the upper surface of the substrate 303 of the “isoelectric focusing protein separation chip” and the lid resin layer 307 is maintained. At that time, it is preferable to adopt a technique in which an external force is applied between the two to maintain a close contact state between the two. As a means for loading an external force applied between the two, a load load application mechanism that is a form in which a load is loaded from the upper surface of the lid 304 can be used. It is desirable to select a form in which the load load application mechanism can disperse a substantially uniform load load over the entire contact surface between the substrate 303 and the lid resin layer 307.

  By maintaining a close contact state, the “solution leakage” in which the sample solution introduced into the flow path 302 leaks out of the flow path from a portion where the upper surface of the substrate 303 and the lower surface of the lid resin layer 307 are in contact is prevented. Therefore, prior to the introduction of the sample solution into the flow path 302, the load application mechanism is operated to maintain a dense contact state. After the completion of the “isoelectric focusing”, by forming an iced body of the “sample solution after electrophoresis” by rapid freezing, it is not necessary to prevent “liquid leakage”. Therefore, after the quick freezing is finished, the external force load using the load application mechanism is finished. As a result, when the operation of peeling the lid 304 is performed after the quick freezing is finished, the upper surface of the lid 304 is in a state where no load is applied.

  In order to stably fix the substrate 303, a form in which the bottom surface of the substrate 303 or the top surface of the substrate 303 is fixed is desirable. For example, a form in which the bottom surface of the substrate 303 is processed into a flat plane and the bottom surface of the substrate 303 is fixed at a predetermined position on a vacuum chuck type fixing stage is preferably used. It is also easy to press and fix the upper surface of the substrate 303, which is preferable. In this case, it is desirable to compress and fix a portion of the upper surface of the substrate 303 where the lid 304 is not provided.

  For the purpose of stably fixing the substrate 303, it is also possible to adopt a form in which the portion with the lid 304 on the upper surface of the substrate 303 is pressed and fixed.

  With the substrate 303 fixed, the lid 304 is peeled off and removed from the substrate 303. Therefore, when the portion of the upper surface of the substrate 303 with the cover 304 is pressed and fixed, and the cover 304 is peeled off, the pressure fixing is released. When the cover 304 is peeled off, the substrate 303 is stably fixed. Difficult to do. In the case of adopting a form in which the portion with the lid 304 on the upper surface of the substrate 303 is pressed and fixed, when the cover 304 is peeled off, a form in which the bottom surface of the substrate 303 is fixed or a portion on the upper surface of the substrate 303 without the cover 304 is compressed. Use a fixed format.

  When peeling / removing the lid 304 from the substrate 303, the substrate 303 is fixed and an external force is applied to one end of the lid 304, so that it is substantially perpendicular to the contact surface between the substrate 303 and the lid 304. Then, one end of the lid 304 is forcibly displaced. Accompanying the displacement of the one end, the lid 304 has a bending structure with respect to the bonding surface. In order to prevent the movement of the substrate 303 when the external force is applied, the substrate 303 is fixed as described above.

  There is no particular limitation on the configuration of the drying mechanism as long as the freeze-dried treatment can be performed on the frozen body of the “sample solution after electrophoresis” while being kept frozen in the flow path. For example, after removing and removing the lid 304 from the substrate 303, the exposed “freeze-up sample solution” frozen substance in the channel. A temperature control mechanism for maintaining a frozen state, a closed tank for sublimating or evaporating the coagulated liquid component (water) contained in the frozen body of the “sample solution after electrophoresis”, and a reduced pressure A freeze-drying mechanism equipped with a mechanism and the like can be mentioned. Using the freeze-drying mechanism, the substrate 303 is placed in a sealed tank, and the inside of the sealed tank is depressurized, whereby the solidified liquid component (water) contained in the frozen body of the “sample solution after electrophoresis” The spots separated along the flow path 302 are dried and fixed as “spots of peptide fragments that have been lyophilized” while maintaining their positions.

  It is preferable that the electrophoresis / drying apparatus compatible with the “isoelectric point protein separation chip” further includes a control unit from the viewpoint of ease of operation. When performing “isoelectric focusing”, the control unit uses the current monitor unit to monitor the current value flowing in the sample solution in the flow path 302 and use it to control the voltage supplied from the power source 402. be able to. Furthermore, the control unit is used to determine the end of “isoelectric focusing” based on the current value monitor, voltage application time, and power consumption, and to control the operation of the cooling mechanism. Can do. Furthermore, the control unit adsorbs the lid 304 or a machine that contacts and fixes the lid 304 to the upper surface of the substrate 303, a mechanism that adsorbs the substrate 303 or contacts and fixes the substrate 303 on the chip base, After the cover 304 is peeled off from the upper surface of the fixed substrate 303, the operation of the moving mechanism that moves away from the lid 304 can be controlled to perform a series of operations for exposing the flow path 302. Furthermore, the control unit controls a pressure reducing / pressurizing mechanism that generates a differential pressure as a solution injection mechanism, and controls a temperature adjusting mechanism and a pressure reducing mechanism as a drying mechanism, or a signal detection unit. As described above, it can be used to confirm the separation state of peptide fragments by “isoelectric focusing”.

  When using an "electrophoresis / drying apparatus" that is compatible with the above "isoelectric point protein separation chip", the method of use will be briefly described below.

  First, the chip 300 is placed in the chip housing portion along the chip guide. The chip accommodating part is composed of a chip table and a sealed tank. The chip 300 is fixed on a chip base. The temperature of the fixed chip 300 is controlled by transferring heat through the chip base and using a temperature control mechanism (temperature sensor, Peltier element, etc.). The temperature control mechanism keeps the chip temperature constant during “isoelectric focusing”, cools the chip during “quick freezing”, heats the chip temperature during “freeze drying”, or “freezes” Used to maintain a temperature below the freezing point in order to maintain the state.

  Next, the lid 304 is pressed against the upper surface of the substrate 303 to fix the lid 304 and maintain a close contact state. A sample solution is injected into one of the reservoirs 305 a and 305 b provided at both ends of the flow path 302. Thereafter, a suction operation or the like is performed to fill the entire channel 302 with the sample solution. Subsequently, catholyte and anolyte are injected into reservoirs 305a and 305b provided at both ends of the channel 302, respectively. All the mechanisms used for the operations such as the above operation, compression and fixing of the lid 304, injection of the sample solution, and the suction operation can be executed by controlling the operation of these mechanisms by the control unit.

  Furthermore, the anode / cathode of the electrode part is disposed so as to be in contact with the anolyte and the catholyte, respectively, through the solution injection through hole of the lid 304. A voltage is applied to the electrode part from the power source through the wiring, and the current value between the anode and the cathode of the electrode part is measured using the current monitor part. Since the current flowing through the sample solution introduced into the flow path 302 gradually decreases from the time of voltage application, the current value or power value is measured, and the change over time is monitored, thereby isoelectric point electricity. The end of separation by electrophoresis can be determined.

  Immediately after the “isoelectric focusing” is finished, the temperature control mechanism is used to cool the chip 300 and freeze the “sample solution after electrophoresis” in the channel 302. In the “sample solution after electrophoresis” in the channel 302, peptide fragments and proteins separated by “isoelectric focusing” are dissolved along the channel 302 by forming spots respectively. It is in a state. The solvent is water, but the acid, base, amphoteric carrier component, etc. used to form the pH gradient in the channel 302 are dissolved, and the temperature at which freezing starts due to the freezing point depression is the freezing point. It is lower than (0 ° C.). Therefore, the entire “sample solution after migration” in the channel 302 is rapidly cooled to a temperature significantly lower than the temperature at which freezing starts, and once in a supercooled state, It is desirable to freeze the entire “sample solution after electrophoresis” at once. That is, the entire flow path 302 is rapidly cooled from the bottom surface of the substrate 303 of the chip 300 in contact with the chip base to a uniform temperature, and the temperature is significantly lower than the temperature at which freezing starts. It is preferable to use a substrate cooling mechanism in a supercooled state, that is, a temperature control mechanism 404. Since this cooling mechanism uniformly cools the entire bottom surface of the substrate 303, it is desirable to adopt an arrangement in which the entire bottom surface of the substrate 303 is in uniform thermal contact, and it is desirable that the chip base and the temperature control mechanism be integrated. .

  Next, the lid 304 is peeled off from the upper surface of the substrate 303. At this time, by continuously cooling the chip 300 in the chip housing portion, the upper surface of the substrate 303 can be exposed while the frozen body of the “sample solution after electrophoresis” is held in the flow path 302.

  In order to bring the upper surface of the substrate 303 and the lower surface of the lid 304 into close contact with each other and release the adhesive force that achieves the adhesive state in a predetermined arrangement, an external force having a direction component substantially perpendicular to the upper surface of the substrate 303 is applied. Is applied to the end of the cover 304, and the lid 304 is bent, and peeling is advanced at a desired speed in a form in which the end of the cover 304 is lifted upward while maintaining the bending at a predetermined curvature. In the process of peeling the lid 304, the peeling progresses rapidly even on the upper surface of the frozen body of the “sample solution after migration” in the groove-like channel 302, which is in contact with the lower surface of the lid resin portion 307 of the lid 304. The iced body of the “sample solution after electrophoresis” is left in the groove-like channel 302, and the peeling of the lid 304 is completed. Thereafter, the separated lid 304 is removed from the upper surface of the substrate 303 by, for example, holding the end portion of the lid 304 chucked by the knob portion and moving the knob portion.

  The frozen body of the “sample solution after electrophoresis” remains in the flow path 302 and is exposed, and the “freeze-drying process” can be performed as it is. After the lid 304 is peeled off, the chip 300 is heated while maintaining the frozen state of the “sample solution after electrophoresis” in a frozen state, or the temperature of the chip 300 is controlled, and the chip housing portion which is a sealed tank is controlled. Perform “freeze-drying treatment” under reduced pressure. When the “freeze-drying process” for removing the coagulated liquid component (water) contained in the frozen sample solution after electrophoresis is completed, each separated spot along the flow path 302 While maintaining the position, it is dried and fixed as a “spot of lyophilized peptide fragments”. After completion of the “freeze drying process”, the temperature of the chip 300 is raised to a temperature exceeding the temperature (dew point temperature) at which water vapor contained in the outside air is condensed, and then the internal pressure of the chip housing portion is returned to atmospheric pressure.

In the “sample pretreatment” step 101, an “electrophoresis / drying device” that conforms to the “isoelectric point protein separation chip” is applied to the “dried product containing the total amount of the prepared peptide fragments” sample according to the above-described procedure. The “isoelectric focusing” step 102 to be used is performed. As a result, a group of peptide fragments obtained by trypsin digesting the ApoB100 protein contained in MDA-LDL contained in the “dried product containing the total amount of the prepared peptide fragments” sample along the flow path 302. And is fixed on the flow path 302 as a group of “freeze-dried peptide fragment spots” while maintaining the spot position.
(2-3) “Isoelectric focusing” conditions A peptide group derived from ApoB100 protein contained in MDA-LDL produced by trypsin digestion, which is the target of “isoelectric focusing”, is the following 6 Broadly divided into two types.
(Type A-0) a peptide fragment having an Arg residue or a Lys residue at the C-terminus and no acidic amino acid residue;
(Type A-1) a peptide fragment having an Arg residue or Lys residue at the C-terminus and containing one acidic amino acid residue;
(Type A-2) a peptide fragment having an Arg residue or Lys residue at the C-terminus and containing two or more acidic amino acid residues;
(Type A′-0) a peptide fragment having an MDA-modified Lys residue at the C-terminus and no acidic amino acid residue;
(Type A′-1) a peptide fragment having an MDA-modified Lys residue at the C-terminus and containing one acidic amino acid residue;
(Type A′-2) a peptide fragment having an MDA-modified Lys residue at the C-terminus and containing two or more acidic amino acid residues;
The pI of these (type A-0) to (type A'-2) peptide fragments is probably (type A-0)> (type A-1)> (type A'-0)> (type A- 2)> (Type A′-1)> (Type A′-2), it is expected to be smaller and at least included in the range of pI3-10.

Therefore, “isoelectric focusing” employs conditions for separating peptide fragments in the pI3-10 range. As an “anolyte”, an alkaline solution, for example, a 40 mM sodium hydroxide aqueous solution (pH 12) is used, and as a “catholyte”, an acid solution, for example, a 100 mM phosphoric acid aqueous solution (about pH 2) is used. Form. The pKa of H ₃ PO ₄ (orthophosphoric acid) is reported as pK _a1 = 2.12, pK _a2 = 7.21, pK _a3 = 12.67 (25 ° C).

  The sample solution of “isoelectric focusing” is the “sample pretreatment” step 101 “dry product containing the total amount of peptide fragments prepared” sample in the following “isoelectric focusing” sample preparation solution. Dissolve in the preparation.

  As the sample preparation solution for “isoelectric focusing”, for example, an aqueous solution containing 66% (v / v) cIEF gel and 3% (v / v) Carrier ampholyte (pI 3-10) is used. In the sample preparation solution, a protein having a known pI and molecular weight M is added as a pI standard, and after “isoelectric focusing”, the pI of the peptide fragment in which spots are formed along the flow path is estimated. When you do this, use it as a standard. For example, 500 fmol / μL angiotensin II (pI 6.74) and 500 fmol / μL fibrinopeptide B (pI 4.0) are added to the sample preparation solution. In addition, if necessary, 2% (v / v) fluorescent markers (3 types of pI4, 5.5, and 9) can be added to the sample preparation solution. The three kinds of fluorescent markers form spots at positions corresponding to pI4, pI5.5, and pI9 along the flow path from the acid side to the alkali side after “isoelectric focusing”. Therefore, from the acid side to the alkali side along the flow path, by observing the fluorescence from the spot of each fluorescent marker with a fluorescence microscope, along the flow path from the acid side to the alkali side, This can be used when determining positions corresponding to pI4, pI5.5, and pI9.

  In the “sample pretreatment” step 101, for example, the ApoB100 protein contained in MDA-LDL contained in 300 μL of serum is fractionated, and the “dried product containing the total amount of the prepared peptide fragments” sample is obtained. It can be dissolved in 8 μL of the prepared solution.

  The total length L of the flow path 302 of the “isoelectric point protein separation chip” is, for example, 35 mm, and the width W of the flow path is, for example, 2 mm. As will be described later, in the “MALDI-TOF MS mass spectrometry” step 103, the total length L of the flow path 302 is divided into 71 sections, and when measuring the MALDI-TOF MS spectrum in each section, the length of each section is measured. The depth ΔL is about 0.5 mm, and the change amount ΔpI of the pI of each section is about 0.1.

The shape, length L _pillar , width W _pillar , and height H _pillar of the columnar body 301 formed on the bottom surface of the channel 302 are usually selected so that L _pillar > H _pillar > W _pillar . The length L _pillar of the columnar body is preferably selected to be 1/10 or less of the length ΔL of each section. The column width W _pillar is preferably selected to be 1/10 or less of the width W of the flow path.

This columnar body is produced at the same time as the flow path 302 is produced. Therefore, the depth D of the flow path 302 is determined by considering the shape of the columnar body formed on the bottom surface of the flow path 302, the length L _pillar , the width W _pillar , and the height H _pillar. It is desirable to select D in the range of 2H _pillar > D ≧ H _pillar . For example, it is more desirable to select the depth D of the flow path 302 so that D = H _pillar . The side wall surface of the columnar body desirably has hydrophilicity. For example, it is preferable to apply a hydrophilic coat on the side wall surface of the columnar body. Further, the columnar bodies formed on the bottom surface portion of the flow path 302 are regularly arranged, and the gap generated between the columnar bodies having hydrophilic side wall surfaces is sufficient to cause capillary action on the sample solution. It is desirable to indicate narrowness.

  When the sample solution is introduced into the channel 302, the sample solution injected into one of the reservoirs wets and spreads on the bottom surface of the hydrophilic channel 302, and further, between the columnar bodies formed on the bottom surface of the channel 302. By proceeding through the narrow gap by utilizing the capillary phenomenon, the entire bottom surface of the flow path 302 can be wetted and spread. Finally, since the sample solution injected into one of the reservoirs enters the entire region where the columnar body 301 is formed on the bottom surface portion in the channel 302, the sample solution is uniformly introduced into the entire channel 302. . Since the sample solution introduction means utilizing this capillary phenomenon is utilized, the sample solution can be uniformly introduced into the entire flow path 302 without using a suction operation.

  After that, the “catholyte” and “anolyte” injected into the reservoirs 305 a and 305 b provided at both ends of the flow path 302 are filled in the flow path 302 at both ends of the sample solution and the flow path 302. It will be in contact.

  When a voltage is applied between “catholyte” and “anolyte” injected into the reservoirs 305a and 305b, a voltage gradient (electric field) is formed along the flow path, and a pH gradient is also formed. The Using the voltage gradient (electric field) and pH gradient formed along the flow path, peptide fragments, proteins, and fluorescent markers contained in the sample solution introduced into the flow path 302 are displayed. “Isoelectric focusing” is performed.

  In “isoelectric focusing”, voltage application between “catholyte” and “anolyte” is, for example, 1.5 kV, 60 s; 0 V, 30 s; 3.0 kV, 720 s. Peptide fragments, proteins, and fluorescent markers contained in the solution are accumulated in the center of the flow path using a voltage gradient (electric field), and then the formed pH gradient and voltage gradient (electric field) are utilized. Then, according to each pI, separation is performed to form a spot.

(3) “MALDI-TOF MS mass spectrometry” step As a group of “spots of peptide fragments that have been freeze-dried”, trypsin digestion of ApoB100 protein contained in MDA-LDL, which is dry-fixed on the channel 302 For a group of peptide fragments obtained by the above, m / z of ionic species [M _peptide + H] ⁺ derived from each peptide fragment (pI, M _peptide ) using “MALDI-TOF MS mass spectrometry”, The flow direction distribution of the ionic strength I (pI, [M _peptide + H] ⁺ ) of the ion species [M _peptide + H] ⁺ is measured.

Specifically, the pI of the peptide fragments (pI, M _peptide ) constituting each spot fixed and dried in the range of pI of 3 to 10 along the flow path 302 from the acid side to the alkali side. Is increasing. Therefore, the ionic species [M _peptide derived from the peptide fragment (pI, M _peptide ) existing in each compartment is divided into 71 compartments along the flow path 302 from the acid side to the alkali side. M / z of + H] ⁺ and its ionic strength I (pI, [M _peptide + H] ⁺ ) are measured.

The peptide fragment (pI, M _peptide ) constituting each spot has an ionic strength I ((M _peptide + H) ⁺ of the ion species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) in a section corresponding to the pI. pI, [M _peptide + H] ⁺ ) shows a maximum, and the ionic strength I (pI, [M _peptide + H] ⁺ ) measured in the front and rear sections of the spot decreases with the distribution of the spot in the channel direction. . The central pI value of each section j is pI _j (j = 1 to 71), and the ion species [M _k + H] ^{+ of} (m / z) = ((M _k +1) / 1) detected in the section j. When the ionic strength I (pI _j , [M _k + H] ⁺ ) is two-dimensionally plotted on the (pI _j , (m / z)) plane, each spot is configured as illustrated in FIG. The ionic species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) is maximal to (pI _mean , (M _peptide +1) / 1) on the (pI _j , (m / z)) plane. It should give an ionic strength distribution showing the points.

In the “MALDI-TOF MS mass spectrometry” step 103, in order to identify a group of “spots of lyophilized peptide fragments” separated along the flow path 302 and retaining the spot position, Dividing into 71 sections from the acid side to the alkali side along 302, the MALDI-TOF MS spectrum is measured in each section j (j = 1 to 71). Based on the MALDI-TOF MS spectrum measured in each section j, (m / z) = ((M _k +1) / 1) of a plurality of ion species k detected in the section j and its ion intensity I (PI _j , [M _k + H] ⁺ ) can be determined.

  Hereinafter, in the “MALDI-TOF MS mass spectrometry” step 103, it is divided into 71 sections along the flow path 302 from the acid side to the alkali side, and in each section j (j = 1 to 71). The procedure for measuring the MALDI-TOF MS spectrum will be described.

  As a group of “spots of peptide fragments that have been lyophilized,” separated on the flow path of the “isoelectric focusing chip” in the “isoelectric focusing” step 102 while retaining the spot position. It is fixed dry. A matrix agent solution used in MALDI-TOF MS measurement is applied to the flow path of the “isoelectric focusing chip” and dried to form a measurement matrix on the flow path.

  After forming the measurement matrix, the MALDI-TOF MS spectrum was sequentially obtained for each of the sections j (j = 1 to 71) divided into 71 sections along the flow path from the acid side to the alkali side. Measure.

  Below, the matrix agent solution used for the measurement of MALDI-TOF MS, the “solution application device” used for application of the matrix agent solution, and the “MALDI-TOF MS mass spectrometer used for measurement of MALDI-TOF MS spectrum” Will be described.

(3-1) “Matrix solution”
The ApoB100 protein itself is a huge protein with more than 4500 amino acids, but the measurement target is a peptide fragment obtained by trypsin digesting the ApoB100 protein contained in MDA-LDL, so the number of amino acids is 50 or less. Yes, as a matrix agent, sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid), CHCA (α-cyano-4-hydroxycinnamic acid), DHB (2,5-dihydroxybenzoic acid), etc. are suitable Used for.

On the other hand, (m / z) = ((M _k +1) / 1 of ionic species [M _k + H] ⁺ derived from peptide fragments existing in each section j (j = 1 to 71) to be measured ) Must be determined with high accuracy. Therefore, in the MALDI-TOF MS spectrum measured in each section j (j = 1 to 71), the ions derived from peptides and proteins having a known mass (M) used for correcting the m / z value. It is preferable to include multiple peaks of species [M + H] ⁺ . For example, as a peptide for mass correction, 900 fmol / μL Bradykinin fragment 1-7 (M = 757.3997), 200 fmol / μL ACTH fragment18-39 (human) (M = 2465.1986), 200 fmol / μL Insulin B chain oxidized (bovine ) (M = 3494.6513).

In “isoelectric focusing”, peptide fragments forming spots are usually electrically neutral. Even at the time of lyophilization treatment, the peptide fragment is usually electrically neutral. After lyophilization treatment, the carboxyl group usually present in the peptide fragment is -COOH, but some of the metal salt type -COO ^- Na ⁺ may be mixed. is there. Prior to mass spectrometry, it is desirable to convert the metal salt form of —COO ⁻ Na ⁺ to the form of —COOH. To that end, a slight amount of trifluoroacetic acid, which serves as a proton donor, is added to the matrix agent solution.

  In addition, after the matrix agent solution is applied, a mixed solvent of water and a polar solvent having a boiling point significantly lower than water and uniformly mixed with water is used in order to easily evaporate the contained solvent.

  For example, 20 μmol / μL sinnamic acid, 0.05% (v / v) TFA, 2% (v / v) formamide, 70% acetonitrile (boiling point 82 ° C.), 900 fmol / μL Bradykinin fragment 1-7 An aqueous solution containing 200 fmol / μL ACTH fragment 18-39 (human) and 200 fmol / μL Insulin B chain oxidized (bovine) can be used.

  When the matrix agent solution having the above composition is used, the coating thickness of the matrix agent solution is set to the depth D of the channel 302 with respect to the channel 302 having a depth D, for example, a channel having a depth of 10 μm. Select a range that does not exceed. By selecting the content concentration of the mass correction peptide and matrix agent described above, it is possible to satisfy the amount of matrix agent per unit area and the amount of added mass correction peptide required for measurement. It is.

  The matrix agent solution is applied in a uniform coating thickness within the flow path of the “isoelectric focusing chip”. The coating thickness of the matrix agent solution is selected in a range that does not exceed the depth D of the flow path. Therefore, the total amount of the matrix agent solution to be applied is selected within a range that does not exceed the total volume of the depression region, as a whole depression region composed of the flow path and the reservoir portions provided at both ends thereof.

(3-2) “Solution coating device”
In order to apply the matrix agent solution with a uniform coating thickness in the flow path, a solution coating apparatus is used. Usually, the solution coating apparatus has a dispenser configuration including a mechanism for pushing out an appropriate amount of the matrix agent solution by a syringe or gas pressure from a fine cylinder and a mechanism for changing the relative position of the fine cylinder and the chip. .

  Using the solution coating apparatus, a predetermined amount of the matrix agent solution is supplied to each of the flow paths, with respect to the entire depression area including the flow paths and the reservoir portions provided at both ends thereof. Since the bottom surface of the flow path is hydrophilic, the supplied matrix agent solution spreads wet so as to uniformly cover the entire bottom surface of the flow path.

The applied matrix agent solution is dried by heating in order to promote the evaporation of the solvent component contained in the matrix agent solution. At that time, the heating temperature T _drying is selected in the range of T _drying <T _bp with respect to the boiling point T _{bp of the} solvent component contained in the matrix agent solution. For example, in the case of the matrix agent solution exemplified above, formaldehyde, acetonitrile, and water are included as solvent components. Among them, the heating temperature T _drying is set to 80 ° C., which is lower than the boiling point 82 ° C. of acetonitrile having the lowest boiling point. Can be set. That is, when the boiling point of the solvent component is exceeded, bubbles are generated and it is avoided that it is difficult to form a uniformly dried matrix.

(3-3) "MALDI-TOF MS mass spectrometer"
In order to specify a group of “spots of lyophilized peptide fragments” that are separated along the flow path and keep the spot position, from the acid side to the alkali side along the flow path, Dividing into 71 sections, MALDI-TOF MS spectra are measured in each section j (j = 1 to 71). Since the change amount ΔpI of pI in each section is about 0.1, the ion species derived from the peptide fragment (pI _mean , M _peptide ) constituting the spot has the “spreading direction σ _pI ” of the spot. Considering this, in addition to the section corresponding to pI _mean , before and after that, a plurality of sections corresponding to the range of pI _mean ± σ _pI are also detected. In addition, there are a plurality of types of peptide fragments (pI _mean , M _peptide ) showing pI _{mean in} the range of pI of each section j. Therefore, the MALDI-TOF MS spectrum measured in each section j includes a peak of the ionic species [M _k + H] ⁺ derived from a considerable number of peptide fragments.

Regarding the peak of the ionic species [M _k + H] ⁺ derived from these considerable number of peptide fragments, the (m / z) = ((M _k +1) / 1) is determined with high accuracy, and the ionic strength I ( pI _j , [M _k + H] ⁺ ) needs to be measured with high accuracy. At least for (m / z) = ((M _k +1) / 1), it is desirable that the accuracy of measurement error Δ (m / z) = 0.2.

For the peptide fragment (pI _mean , M _peptide ) constituting the spot, based on the distribution of ionic strength I (pI, [M _peptide + H] ⁺ ) caused by the “spreading direction σ _pI ” of the spot, When specifying the pI _mean of the center of the spot, it is necessary to compare the ionic strength I (pI _j , [M _k + H] ⁺ ) measured in the adjacent section. In order to compare the peak intensities between the spectra measured in each compartment, the spectra measured in each compartment are based on the ionic strength of the ionic species derived from the internal standard peptide added to the matrix solution. Standardization of peak intensity is performed. It is desirable to use one of the above-described peptides for mass correction as an internal standard peptide used for standardization of the peak intensity. For example, in the case of the matrix agent solution exemplified above, among the mass correction peptides, the addition amount of Bradykinin fragment 1-7 is set high, and it is also used as an internal standard peptide used for peak intensity standardization. .

The “MALDI-TOF MS mass spectrometer” used for measurement is divided into a target holder for setting the chip in the mass spectrometer and a laser beam irradiation position along the flow path on the chip. A light irradiation mechanism for moving and irradiating laser light, and an analysis mechanism for obtaining a mass spectrum by detecting ion species [M _k + H] ⁺ derived from peptide fragments that are ionized by laser light irradiation to each section It is.

The laser beam irradiation spot diameter d _laser is selected in the range of 50 μm to 200 μm. The wavelength of the pulse laser beam used in “MALDI-TOF MS” is selected according to the type of matrix agent to be used. By repeating pulsed laser light irradiation and integrating the detected ion intensity of the ion species [M _k + H] ⁺ , the mass spectrum of each section is measured. The number of repetitions of pulsed laser light irradiation is selected so that the peak intensity of the ion species caused by the internal standard peptide used for standardizing the peak intensity falls within a predetermined intensity range.

The range of (m / z) of the ion species [M _k + H] ⁺ to be measured is within the range of 400 <(m / z) <15000 because the target peptide fragment is limited to peptide fragments having 5 or more amino acids. Set.

For example, (m / z) is corrected based on the peak position of the ion species [M _{Mass-standard} + H] ⁺ derived from the above-mentioned three kinds of peptides for mass correction on the measured spectrum. Further, among the peptides for mass correction measured in each section j, they are derived from peptide fragments based on the peak intensity I _j (Bradykinin) of ionic species [M _Bradykinin + H] ⁺ derived from Bradykinin fragment 1-7. The peak intensity I (pI _j , [M _k + H] ⁺ ) of the ion species [M _k + H] ⁺ is standardized and the relative value (I (pI _j , [M _k + H] ⁺ ) / I _j (Bradykinin)) × 100≡I _rel. (PI _j , [M _k + H] ⁺ ) is used to compare spectra of different sections.

(4) “Sample profiling” step 104
In the “sample profiling” step 104, along the flow path, the “isoelectric focusing” is performed based on the MALDI-TOF MS spectrum divided into 71 sections and measured in each section j (j = 1 to 71). In the “electrophoresis” step, the pI _mean and the molecular weight M _peptide of the peptide fragments (pI _mean , M _peptide ) forming each spot are identified.

(4-1) Estimation of pI value (pI _j ) of each section j First, the section j is divided into 71 sections along the flow path, and each section j (j = 1 to 71) When measuring the MALDI-TOF MS spectrum, the laser light irradiation position, the center position of the section j (j = 1 to 71), and the pI value (pI _j ) of L _mean ( _j ) are estimated.

When “isoelectric focusing” is performed, the fluorescent markers (three types of pI4, 5.5, and 9) added to the sample solution are added to the flow path when “isoelectric focusing” is completed. Along the lines, spots are formed at positions corresponding to pI4, 5.5 and 9, respectively. At the time when the “freeze-drying process” is completed, the position where the fluorescence caused by these fluorescent markers is emitted is specified using a fluorescence microscope. The center position of each fluorescent marker spot, L (pI = 4), L (pI = 5.5), L (pI = 9) is the center position of each section j (j = 1 to 71), L _mean ( j) Specify which of them matches. The central position of the section j and the pI value (pI _j ) of L _mean (j), which coincide with L (pI = 4), L (pI = 5.5), and L (pI = 9), respectively. , _{respectively, pI j ≒ 4, pI j} ≒ 5.5, a pI _j ≒ 9.

In addition, when pI is estimated, a spot of pI standard peptide, angiotensin II (pI6.74, Mangiotensin _II = 1045.534), fibrinopeptide B (pI4.0, M _{fibrinopeptide B} = 1566.969) used for the reference is estimated. Is identified. Was determined from the spot position of the fluorescent _{marker, pI j ≒ 4, pI j} ≒ 5.5, based on the respective section of pI _j ≒ 9, estimates the compartment center of the spot of the two pI standard peptide is located . The peak (m / z) = (M _angiotensin ) of the ion species ([M _{angiotensin II} + H] ⁺ ) derived from angiotensin II in the MALDI-TOF MS spectrum measured in the front and rear five sections centering on the estimated section _II + 1) and the peak (m / z) = (M _{fibrinopeptide B} + 1) of the ion species [M _{fibrinopeptide B} + H] ⁺ ) derived from _{fibrinopeptide B} are identified.

The peak intensity relative value I _rel. (PI _j , [M _{angiotensin II} + H] ⁺ ) of the ionic species [M _{angiotensin II} + H] ⁺ ) derived from the specified _{angiotensin II} is compared _. One section can be found where pI _j , [M _{angiotensin II} + H] ⁺ ) shows the maximum. The pI value (pI _j ) of L _mean (j), the central position of the section j where the spot center of angiotensin II is found, is pI _j ≈6.74. _Comparing the peak intensity relative value I _rel. ( _{PI j} , [M _{fibrinopeptide B} + H] ⁺ ) of the ionic species [M _{fibrinopeptide B} + H] ⁺ ) derived from the identified fibrinopeptide B, the peak intensity relative value I _rel. It is possible to find one section where pI _j , [M _{fibrinopeptide B} + H] ⁺ ) shows a maximum. is found spot center of fibrinopeptide B, the center position, pI values of L _mean (j) of the compartment j (pI _j) is a pI _j ≒ 4.0.

The central position of the compartments of the two ends of the channel, i.e., pI values of the compartments 1 of the center position _{L mean (1) (pI 1} ), pI value of compartments 1 of the center position _{L mean (71) (pI 71} ) is PI ₁ ≈2 and pI ₇₁ ≈12 from “catholyte”, for example, about pH 2 of 100 mM phosphoric acid aqueous solution, and “anolyte”, for example, pH 12 of 40 mM sodium hydroxide aqueous solution.

Through the above operation, the central position of the seven sections in total, the pI value (pI _j ) of L _mean ( _j ) is specified. Along the channel, is divided, the center position of each partition j, pI values of _{L mean (j) (pI j} ) , the central position of the total of seven compartments identified, pI of L _mean (j) Based on the value (pI _j ), it is estimated by interpolation. The error of the estimated center position of each section j, pI value (pI _j ) of L _mean (j) is about ± 0.1.

  The sample solution for performing “isoelectric focusing” contains a peptide fragment group derived from anti-MDA-LDL antibody, a peptide fragment group derived from trypsin, and trypsin. Since the pI and molecular weight M of the peptide fragment group derived from anti-MDA-LDL antibody, the peptide fragment group derived from trypsin, and trypsin are known in advance, the spot position and the peak position of the ionic species (m / z ) Is also identified using techniques applied to the pI standard peptide.

As described above, the peak intensity relative value I _rel. (PI _j , [M _k + H] ⁺ ) is basically the same for any sample solution.

(4-2) Determination of (pI _mean , M _peptide ) of the ApoB100 protein-derived peptide fragment group contained in MDA-LDL Next, the segment is divided into 71 sections along the flow path, and each section j ( j = 1-71), the peak of ionic species derived from the ApoB100 protein-derived peptide fragment group contained in MDA-LDL, which is generated by trypsin digestion, is extracted from the MALDI-TOF MS spectrum. The procedure for specifying the pI _mean at the center of the spot formed by the peptide fragment will be described. That is, the procedure for determining the peptide fragment (pI _mean , M _peptide ) for the peptide fragment group derived from ApoB100 protein contained in MDA-LDL, which is generated by trypsin digestion, will be described.

(4-2-1) Extraction of peak of ion species in MALDI-TOF MS spectrum Concentration of peptide fragment derived from ApoB100 protein contained in sample solution and generated by trypsin digestion in MDA-LDL In some cases, the peak intensity of ionic species derived from peptide fragments having a low concentration is low. Since the peak of the ion species having a small peak intensity is extracted from the MALDI-TOF MS spectrum measured in each section j (j = 1 to 71), the measured spectrum is smoothed.

The MALDI-TOF MS spectrum is actually obtained by counting the number N (t _i ) of ion species that reached the detection system between the flight times t _i and t _i + δt (time window width δt). Therefore, in practice, it is a discrete data string of the form (t _i , N (t _i )). δt is set sufficiently narrow with respect to the increment Δt _{i of} t _i . The number N _m (t _i ) of ion species that are ionized by each laser light pulse and reach the detection system during this time window width δt follows a Poisson distribution with a center value N _mean (t _i ). ΣN _m (t _i ) = N (t _i ), in which the number of counts N _m (t _i ) of each laser light pulse (m) is integrated, ΣN _m (t _i ) = N (t _i ) As the value increases, it _approaches m · N _mean (t _i ). The degree of variation in the Poisson distribution is about (N _mean (t _i )) ^{1/2 with} respect to the center value N _mean (t _i ), and (N _mean (t _i )) ^1/2 / N _mean ( t _i ) increases significantly as the central value N _mean (t _i ) decreases. Therefore, in the case of a peak of an ion species having a small peak intensity, ΣN _m (t _i ) does not show a maximum value at the original peak center position (t _i-peak ), and the point showing the maximum appears to be slightly shifted. There is. By performing smoothing of the measured spectrum, the original peak center position (t _i-peak ) can be estimated.

  In addition, when counting the number of ion species that have reached the detection system, noise components are present, and the peak of an ion species having a small peak intensity is originally measured with a relatively low S / N ratio. By performing smoothing of the spectrum, it is possible to relatively reduce the influence of noise.

The smoothing of the measured spectrum is in principle equivalent to taking a moving average over a discrete data sequence of the form (t _i , N (t _i )).

In practice, the mass spectrum is smoothed by taking the average of the spectrum m / z and the signal values (t _i , N (t _i )) for each specified number using the Savitzky-Golay filter. FIG. 7 shows an example of the result of smoothing the measured spectrum.

After the smoothing process, (ti _-peak-obs , N (ti _-peak-obs )) showing the maximum is selected and used as the measured value of (m / z) of the peak. With the smoothing process, the measured value of the peak (m / z) is about ± 0.2 systematic error with respect to the original peak center (t _i-peak , N (t _i-peak )). It may indicate δ (m / z). On the other hand, the difference in (m / z) between peaks observed on the same spectrum is ± 1 at the minimum, so adjacent peaks are sufficiently separated.

  When comparing spectra measured in adjacent compartments, the peaks observed on the two spectra with the same (m / z) within the error range are ionic species derived from the same peptide fragment. It is necessary to determine whether or not.

Peptide fragments (pI, M _peptide ) separated along the flow path by “isoelectric focusing” form a spot indicating “spreading direction σ _pI ” centered on pI _mean. ing. The distribution of the amount of peptide fragments (pI, M _peptide ) in the spot can be regarded as a Gaussian distribution centering on pI _mean and indicating “spreading direction σ _pI ”. Thus, the ionic strength _{I (pI, [M peptide +} H] +) ion species [M _peptide ⁺ H] + derived peptide fragments (pI, M _peptide) Measurement of the, around the pI _mean, "the flow path direction Intensity change like Gaussian distribution showing “spread σ _pI ” is shown.

The peak intensity relative value I _rel. (PI _j , (m / z) _k ) of the peak (pI _j , (m / z) _k ) found on the spectrum measured in each section is expressed as (pI _j , ( m / z)) When two-dimensionally plotting on the plane, the peak intensity relative value I _rel. (pI, [ _p ] of the ion species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) forming the spot M _k + H] ⁺ ) indicates a Gaussian distribution-like intensity change. Considering that the measured value of peak (m / z) shows a systematic error of about ± 0.2 with the smoothing process, the measured value of peak (m / z) _k is within ± 0.2. In which the peak intensity relative value I _rel. (PI _j , (m / z) _k ) changes in intensity like a Gaussian distribution is an ionic species derived from the same peptide fragment (pI, M _peptide ) [ M _peptide + H] ⁺ peak.

In FIG. 8, the peak intensity relative value I _rel. (PI) of the peak (pI _j , (m / z) _k ) two-dimensionally plotted on the (pI _j , (m / z)) plane in accordance with the above judgment criteria _. Based on the distribution of _j , (m / z) _k ), an example is shown in which the peak of the ionic species [M _peptide + H] ⁺ derived from the same peptide fragment (pI, M _peptide ) is specified.

At this time, the “span in the flow path direction σ _pI ” of each spot has substantially the same degree of “ _{span in the} flow path direction σ _pI ” when the center value pI _mean is substantially the same. Standard "flow path direction of the spread sigma _pI" is, in each pI regions, respectively, a fluorescent marker, approximately equal to the "flow path direction of the spread sigma _pI" found in spot pI standard peptide.

For example, equal molecular weight M _peptide, the difference? PI _mean the pI _mean is, for the "spread of the channel direction sigma _pI" is ΔpI _mean ≒ σ _pI, two peptide fragments _{(pI mean, M peptide),} ( When pI _mean + ΔpI _mean , M _peptide ) is separated, the peak intensity relative value I _rel. (pI _j , M _peptide +1) of the peak of (m / z) = (M _peptide +1) found in this pI region ) Indicates a spot having an abnormally wide “spread direction σ _pI ”. Even in such a case, in this pI region, the two peptide fragments can be separated by separating them as an overlap of Gaussian distributions having two central values and having a standard “spreading direction σ _pI ”. It is possible to extract peaks of ion species derived from (pI _mean , M _peptide ) and (pI _mean + ΔpI _mean , M _peptide ).

In other words, assuming a Gaussian distribution with a standard “span in channel direction σ _pI ” in each pI region, a two-dimensional plot is made on the (pI _j , (m / z)) plane. , peak _{(pI j, (m / z} ) k) of the peak intensity relative value _{I rel. (pI j, (} m / z) k) by analyzing the distribution of a single peptide fragment (pI, M _peptide ) Form a spot, or coincidentally, the molecular weights coincide, and it can be determined whether spots of peptide fragments slightly different in pI _mean overlap.

However, if the molecular weight M _peptide coincides and the difference ΔpI _{mean of} pI _mean is 0.1 ≧ ΔpI _mean , whether or not spots of a plurality of peptide fragments overlap in the above determination procedure It is impossible to determine whether or not.

When the above-described “peak extraction” process is performed, a peptide fragment (pI, M _peptide ) derived from the ApoB100 protein contained in MDA-LDL produced by digestion of trypsin that has been separated along the flow path. The pI _{mean at the} center of the spot and the peak intensity relative value I _rel. (PI, [M _k + H] ⁺ ) of the ion species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) are determined.

At that time, the specific error of pI _{mean at} the center of the spot of the peptide fragment (pI, M _peptide ) does not exceed the standard “spreading direction σ _pI ” in the pI region, but the error δpI _mean Is about ± 1/2 (σ _pI ). Therefore, the error δpI _mean is ± 0.4 at the maximum.

(4-2-2) Matching (identification) of extracted peaks among multiple serum samples
In order to compare the amount of peptide fragments (pI, M _peptide ) derived from ApoB100 protein contained in MDA-LDL produced by trypsin digestion between multiple serum samples, a sample solution prepared from each serum sample was used. “Isoelectric focusing” and “MALDI-TOF MS mass spectrometry” are performed under the same conditions. The same peptide was used for the peptide fragments (pI _mean , M _k ) that were subjected to “isoelectric focusing” of the sample solution prepared from each serum sample, and “peak extraction” was performed along each channel. Whether or not it is a fragment (pI, M _peptide ) is associated (identified).

In the peptide fragment (pI _mean , M _k ) for which “peak extraction” has been performed along each flow path according to the above procedure, pI _{j of} each section j is estimated by interpolation, estimate of pI _j has an error? pI _i of about ± 0.1, the error? pI _mean specific time of pI _mean of spot center, the maximum, has a ± 0.4. Therefore, for the same peptide fragment (pI, M _peptide ) for which “peak extraction” has been performed between any two flow paths, when the pI _mean specific results at the center of the spot are compared, the estimated pI _{mean of} both The “apparent difference” may reach a maximum, δpI _i + δpI _mean , ± 0.5. On the other hand, considering that the systematic error δ (m / z) associated with the smoothing process is ± 0.2 at the maximum, the “apparent difference” between the estimated values of the molecular weights M _k is the maximum, ± May reach 0.3.

Therefore, the peptide fragment (pI _mean , M _k ) separated between the two arbitrary flow paths along the flow path and subjected to “peak extraction”, respectively, has the above-mentioned pI _mean estimated value “ Considering the “apparent difference” and the “apparent difference” of the estimated value of the molecular weight M _k , whether or not they are the same peptide fragment (pI, M _peptide ) is associated (identified).

The relative order in the flow path of the spots of peptide fragments (pI, M _peptide ) separated by “isoelectric focusing” follows the original order of pI. The order will never be reversed. Therefore, it is possible to determine the correspondence relationship for the peptide fragment (pI _mean , M _k ) for which “peak extraction” has been performed, according to the relative order of (pI _mean , M _k ) in the flow path.

Specifically, along the flow path, the range of pI3 to pI10 is set to the center position of each fluorescent marker spot, L (pI = 4), L (pI = 5.5), L (pI = 9). ), PI standard peptide, angiotensin II (pI 6.74), fibrinopeptide B (pI 4.0) spot central position, L (pI = 6.74), L (pI = 4.0), 5 regions ( Divide into 3). For each channel, the relative order of (pI _mean , M _k ) is determined for a group of peptide fragments (pI _mean , M _k ) for which “peak extraction” has been performed in each divided region.

In the case of the same peptide fragment (pI, M _peptide ), considering the condition that the “apparent difference” of the estimated value of the molecular weight M _k is within ± 0.3 (pI _mean , M _k ) in accordance with the relative order, whether the peptide fragment (pI _mean , M _k ) for which “peak extraction” has been performed between the two flow paths is the same peptide fragment (pI, M _peptide ), or not The association (identification) is advanced.

When it is associated (identified) with the same peptide fragment (pI, M _peptide ), it is derived from the peptide fragment (pI _mean , M _k ) that has undergone “peak extraction” along two flow paths. The maximum point coordinates (pI _mean , (m / z) _k ) of the peak intensity relative value I _rel. (PI _j , (m / z) _k ) of the ion species are respectively (pI _j , (m / z)) When two-dimensional plotting is performed on a plane, a maximum point is obtained on the two two-dimensional plots within the range of systematic error δpI _i associated with pI _j and systematic error δ (m / z) associated with smoothing processing. The coordinates (pI _mean , (m / z) _k ) correspond.

The corresponding local maximum coordinates (pI _mean , (m / z) _k ) can be seen within the systematic error δpI _i associated with the estimation of pI _j and the systematic error δ (m / z) associated with the smoothing process. The peptide fragment (pI _mean , M _k ) that is not released is determined to be a _peptide fragment (pI, M _peptide ) contained only in one sample solution.

  Provided by multiple individuals to screen for peptide fragments that reflect differences in the degree of oxidation of ApoB100 protein contained in sorted MDA-LDL from blood samples taken from subjects. With respect to the ApoB100 protein sample contained in the MDA-LDL fractionated from the blood sample, the extracted peaks are associated (identified) among a plurality of serum samples by the above procedure.

(5) “Data analysis / marker identification” step A sample of ApoB100 protein contained in the MDA-LDL separated from the blood sample provided by each individual is generated by trypsin digestion according to the above procedure. The peak intensity relative to the ionic species derived from the peptide fragment (pI _mean , M _k ) that is associated (identified) to correspond to the peptide fragment (pI, M _peptide ) derived from the ApoB100 protein contained in MDA-LDL The value I _rel. (PI _j , (m / z) _k ) is a peptide fragment (pI, M _peptide ) derived from the ApoB100 protein contained in MDA-LDL, generated by trypsin digestion, identified in the individual. _Is the peak intensity relative value I _rel. (PI, M _peptide +1) of the ion species [M _peptide + H] ⁺ derived from.

For each individual, the maximal point coordinates (pI _mean , M) of the peak intensity relative value I _rel. (PI _j , (m / z) _k ) of the ionic species derived from the identified peptide fragment (pI _mean , M _k ) As described above, (m / z) _k ) includes the systematic error δpI _i associated with the estimation of pI _j and the systematic error δ (m / z) associated with the smoothing process. Therefore, for a plurality of individuals, specifically, a group of 10 patients diagnosed with ischemic heart disease (number of coronary artery lesion branches: 3) and a non-patient group (number of coronary artery lesion branches: 0) of 23, a total of 33 people The maximum point coordinates (pI _mean , (m / z) of the peak intensity relative value I _rel. (PI _j , (m / z) _k ) of the ion species from which the identified peptide fragment (pI _mean , M _k ) is derived _k ) is averaged, and the average value (pI _mean , (m / z) _k ) _Av. is assigned to the pI of the associated (identified) peptide fragment and the ionic species derived from the peptide fragment [M _peptide + H ] ⁺ (M / z) = (M _peptide +1).

For each of the 33 individuals, the peak intensity relative value I _rel. (PI, M _peptide +1) of the ionic species derived from the associated (identified) peptide fragment (pI, M _peptide ) and the maximum point coordinates A list of (pI, M _peptide +1) is created. Peptide fragments (pI, M _peptide ) listed in this list are present in the sample solution for any of the 33 individuals, apart from the peak intensity relative value I _rel. (PI, M _peptide +1) _. It has been confirmed that. Of course, the peptide fragments (pI, M _peptide ) described in this list are generated by trypsin digesting the ApoB100 protein contained in the MDA-LDL to be separated from the blood sample provided by each individual. Peptide fragment.

  In addition, between 10 patients in the patient group (3 coronary artery lesion branches) and 23 non-patient group (0 in the coronary artery lesion branches), the progress of oxidation in the ApoB100 protein contained in the sorted MDA-LDL Are clearly different, the “progression of oxidation” is high in 10 patient groups (3 coronary lesion branches), and the “progression of oxidation” in 23 non-patient groups (0 coronary lesion branches). Is low.

On the other hand, since the amount of ApoB100 protein contained in MDA-LDL, which is fractionated from blood samples provided by a total of 33 people, differs depending on each individual, the peak intensity described in the above list is included. The relative value I _rel. (PI, M _peptide +1) cannot be directly compared. The peak intensity relative value I _rel. (PI, M _peptide +1) described in the list of each individual is included in MDA-LDL separated from the serum sample used for the preparation of the sample solution. It is necessary to normalize for the amount of protein.

When preparing a sample solution, the amount (molar amount) of the peptide fragment produced by trypsin digesting ApoB100 protein contained in MDA-LDL is the amount of ApoB100 protein contained in MDA-LDL used. It is proportional to (molar amount). Therefore, when all the peptide fragments (pI, M _peptide ) described in the list are averaged in the amount (molar amount) of the peptide fragment, the average value of the ApoB100 protein contained in the used MDA-LDL is also calculated. It is proportional to the amount (molar amount). Of course, the peak intensity relative value I _rel. (PI, M _peptide +1) described in the list is proportional to the amount (molar amount) of the peptide fragment (pI, M _peptide ).

On the other hand, the ion species derived from the peptide fragments _{(pI, M peptide) [M} peptide + H] + peak intensity _{I (pI, [M peptide +} H] +) , the peptide fragment in the sample solution of (pI, M _peptide) Concentration; using C (pI, M _peptide ) and the production efficiency (ionization efficiency) p (pI, M _peptide ) of the ionic species [M _peptide + H] ⁺ , I (pI, [M _peptide + H] ⁺ ) = p (pI, M _peptide ) · C (pI, M _peptide ).

  An analysis procedure that can be suitably used in the “data analysis / marker identification” step 105 will be described below.

Correspondence (identification) is made peptide fragment (pI, M _peptide) for a group of, for each peptide fragment (pI, M _peptide), the average value _{(pI mean, (m / z} ) k) a _Av. The calculated and matched (identified) pI of the peptide fragment and (m / z) = (M _peptide +1) of the ion species [M _peptide + H] ⁺ derived from the peptide fragment are used.

On the other hand, the peak intensity relative value I _rel. (PI, M _peptide +1) described in each individual list is an ion derived from Bradykinin fragment 1-7 among the peptides for mass correction measured in each section j. Based on the peak intensity I _j (Bradykinin) of the species [M _Bradykinin + H] ⁺ , the peak intensity I (pI _j , [M _k + H] ⁺ ) of the ionic species [M _k + H] ⁺ derived from the peptide fragment is standardized. , Relative value (I (pI _j , [M _k + H] ⁺ ) / I _j (Bradykinin)) × 100≡I _rel. (PI _j , [M _k + H] ⁺ ).

The peak intensity relative value I _rel. (PI, M _peptide +1) described in the list of each individual is the “standardized peak intensity” subjected to the above-mentioned standardization process, but in a blood sample provided from a total of 33 persons In order to make a quantitative comparison in, according to the following procedures (5-1), (5-2), described in the list of each individual Sm (Sm = 1 to 33), "standardized peak intensity" I _rel. (PI, M _peptide +1) _Sm is subjected to “logarithmic transformation” processing and “normalization” processing to calculate “normalized peak intensity” I (pI, M _peptide +1) _normal-Sm .

(5-1) “Logarithmic transformation” process “Standardized peak intensity” I _rel. (PI, M _peptide +1) _Sm logarithmically converted in the list of each individual Sm (Sm = 1 to 33) Logarithm normalized peak intensity “ln (I _rel. (PI, M _peptide +1) _Sm )

Concentration of peptide fragment (pI, M _peptide ) in the sample solution of each flow path; C (pI, M _peptide ) _Sm and production efficiency (ionization efficiency) p (pI, M) of the ionic species [M _peptide + H] ⁺ _peptide ) can be expressed as I (pI, [M _peptide + H] ⁺ ) _Sm = p (pI, M _peptide ) · C (pI, M _peptide ) _Sm The generation efficiency (ionization efficiency) p (pI, M _peptide ) of the ion species [M _peptide + H] ⁺ is essentially the same for the same ion species. In addition, the peak intensity I _j (Bradykinin) of the ion species [M _Bradykinin + H] ⁺ derived from Bradykinin fragment 1-7 measured in each channel is basically equal.

Therefore, “log normalized peak intensity” ln (I _rel. (PI, M _peptide +1) _Sm ) = ln (C (pI, M _peptide ) _Sm ) + ln (p (pI, M _peptide )) − ln (I _j (Bradykinin)), {ln (p (pI, M _peptide ))-ln (I _j (Bradykinin))} should be equal in any channel.

(5-2) “Normalization” process First, for each individual Sm (Sm = 1 to 33), in a group (N _peak type) of peptide fragments (pI, M _peptide ) associated (identified), “ “Average value” of log-normalized peak intensity ”ln (I _rel. (PI, M _peptide +1) _Sm ), ln (I _rel. ( _PI , M _peptide +1)) _av.-Sm = (1 / N _peak ) Σln (I _rel. (PI, M _peptide +1) _Sm ) and “standard deviation: σ (ln (I _rel. (PI, M _peptide +1))) _Sm ” relative to the “average value” are calculated.

The “average value” is ln (I _rel. ( _PI , M _peptide +1)) _av.-Sm = (1 / N _peak ) Σln (I _rel. (PI, M _peptide +1) _Sm ) = {(1 / N _peak ) Σln (C (pI, M _peptide ) _Sm )} + {(1 / N _peak ) Σln (p (pI, M _peptide ))}-ln (I _j (Bradykinin)). Among them, the part {(1 / N _peak ) Σln (p (pI, M _peptide ))}-ln (I _j (Bradykinin)) should be equal in any channel.

For each individual Sm (Sm = 1 to 33), the “normalized peak intensity” I (pI, M _peptide +1) _normal-Sm of each peptide fragment (pI, M _peptide ) associated (identified) is I (PI, M _peptide +1) _normal-Sm = [(ln (I _rel. (PI, M _peptide +1) _Sm ) -ln (I _rel. ( _PI , M _peptide +1)) _av.-Sm ) / σ (ln (I _rel. (PI, M _peptide +1))) _Sm ].

(5-3) “Z scoring” processing For each individual Sm (Sm = 1 to 33), for a group (N _peak type) of peptide fragments (pI, M _peptide ) associated (identified), each "normalized peak intensity" I (pI, M _peptide +1) after calculating the _normal-Sm "normalized peak intensity" of each peptide fragment _{(pI, M peptide) I (} pI, M peptide +1) normal- _Sm is subjected to a “Z score” process according to the following procedure.

A total of 33 “normalized peak intensities” I (pI, M _peptide +1) _normal-Sm of each peptide fragment (pI, M _peptide ) calculated for each individual Sm (Sm = 1 to 33) Among all sample groups, consisting of samples of individual individuals Sm (Sm = 1 to 33), the “average value among all sample groups”; I (pI, M _peptide +1) _normal-av. = (1/33 ) ΣI (pI, M _peptide +1) _normal-Sm and “standard deviation in all sample groups: σ (I (pI, M _peptide +1) _normal )” with respect to the “average value in all sample groups” To do.

Then. The calculated peptide fragment (pI, M _peptide ) for each individual Sm (Sm = 1 to 33) using the calculated “average value in all sample groups” and “standard deviation in all sample groups”. ) “Normalized peak intensity: I (pI, M _peptide +1) _normal-Sm ” [I (pI, M _peptide +1) _normal-Sm ) −I (pI, M _peptide +1) _normal-av. ) / σ (I (pI, M _peptide +1) _nprmal )] is calculated and used as the Z value (I (pI, M _peptide +1) _Z-score-Sm ).

Using the Z value (I (pI, M _peptide + 1) _Z-score-Sm ) of each peptide fragment (pI, M _peptide ) calculated for each individual Sm (Sm = 1 to 33), The average value of the calculated “Z value: I (pI, M _peptide +1) _Z-score-Sm ” was calculated for 10 samples of the disease group (3 coronary artery lesion branches), and the disease group (3 coronary artery lesion branches 3) was calculated. ) The average value of the calculated “Z value: I (pI, M _peptide +1) _Z-score-Sm ” in a subgroup of 10 samples is at least 0.60 or more, preferably 0.70 or more Select “peak”.

The “peaks” selected based on the above criteria are “normalized peaks” in 10 samples in the disease group (3 coronary lesion branches) compared to 23 samples in the control (non-patient) group (0 coronary lesion branches). Intensity: I (pI, M _peptide + 1) _normal-Sm ”corresponds to a statistically significant“ peak ”. Actually, since the total of the “Z value” for all 33 samples is 0, the calculated “Z value: I in the subgroup of the control (non-patient) group (coronary artery lesion branch number 0) 23 samples”. The average value of “(pI, M _peptide + 1) _Z-score-Sm ” is a more negative value than − (0.65) × (10/23) ≈−0.28. Therefore, the average value of the calculated “Z value: I (pI, M _peptide +1) _Z-score-Sm ” in 10 samples of the disease group (number of coronary artery lesion branches 3) and the control (non-patient) group (coronary artery lesion) The difference from the calculated “Z value: I (pI, M _peptide +1) _Z-score-Sm ” in the subgroup of 23 samples is at least “standard deviation in all sample groups: It is 0.93 or more of “σ (I (pI, M _peptide +1) _normal )”, and it can be considered that there is a statistically significant difference.

Using the Z value (I (pI, M _peptide + 1) _Z-score-Sm ) of each peptide fragment (pI, M _peptide ) calculated for each individual Sm (Sm = 1 to 33), Perform receiver operating characteristic (ROC) analysis. The Z value (I (pI, M _peptide + 1) _Z-score-Sm ) of the peptide fragment (pI, M _peptide ) calculated from the measurement result of the sample derived from each individual Sm (Sm = 1 to 33) is large. Arranged in order, "ranking" from 1st to 33rd is made.

Disease group (coronary lesions branch number 3) 10 in a sample, Z value _{(I (pI, M peptide +1} ) Z-score-Sm) ratio is the number of samples is within the upper N ^th position (True positives rate or Sensitivity): TPR (pI, M _peptide +1: N ^th );
Control (non-patient) group (coronary lesions branch number 0) 23 in a sample, Z value _{(I (pI, M peptide +1} ) Z-score-Sm) ratio is the number of samples is within the upper N ^th position (False positives rate ): FPR (pI, M _peptide +1: N ^th );
Control (non-patient) group (coronary artery lesions branch number 0) 23 in the sample, Z value _{(I (pI, M peptide +1} ) Z-score-Sm) is the ratio of the number of samples that do not contain within the upper N ^th position (True negative rate or Specificity), TNR (pI, M _peptide +1: N ^th );
The above three probabilities are calculated. Of course, FPR (pI, M _peptide + 1: N ^th ) + TNR (pI, M _peptide + 1: N ^th ) = 1.

Calculated on the ROC space, where the X-axis is TNR and the Y-axis is TPR (TNR (pI, M _peptide +1: N ^th ), TPR (pI, M _peptide +1: N ^th )) (N ^th = 1 ~ 33) are plotted. On the ROC space represented by (TNR, TPR), an ROC curve passing through (0, 1) and (1, 0) and the 33 plot points is drawn. The area A _plot of the region sandwiched between the drawn ROC curve and the X axis (Specificity) is calculated by numerical integration. The calculated area A _plot corresponds to AUC “area under the ROC curve”. Based on the criterion that the “AUC value” calculated by the ROC analysis is at least 0.60 or more, preferably 0.70 or more, a disease group (number of coronary artery lesion branches 3) and a control (non-patient) Peptide fragments (pI, M _peptide ) effective for estimating the group (number of coronary artery lesion branches 0) can be selected.

The total of 33 samples are composed of 10 samples of the disease group (number of coronary artery lesion branches 3) and 23 samples of the control (non-patient) group (number of coronary artery lesion branches 0), so that N ^th = 10 When the plot points (TNR (pI, M _peptide +1: 10), TPR (pI, M _peptide +1: 10)) match (1, 1) corresponding to perfect prediction in the ROC space, the “AUC value” is Furthermore, the average value of the calculated “Z value: I (pI, M _peptide +1) _Z-score-Sm ” in 10 samples of disease group (number of coronary artery lesion branches 3) is at least 0.65 Based on the above criteria, the selected “peaks” are also peptide fragments (pI, M) that are effective in estimating the disease group (3 coronary lesion branches) and the control (non-patient) group (0 coronary lesion branches). _peptide )).

Based on the selection criteria based on the “AUC value” calculated by the above ROC analysis, “peaks” whose “AUC value” is calculated to be at least 0.60 or more are selected. For the selected “peak”, the amino acid sequence of the _peptide fragment (pI, M _peptide ) produced by trypsin digestion from the ApoB100 protein contained in MDA-LDL that gives the “peak” is specified.

(5-4) Identification of amino acid sequence of _peptide fragment (pI, M _peptide ) that gives selected “peak” The selected “peak” is trypsin digestion of ApoB100 protein contained in MDA-LDL. Derived from the peptide fragment produced in The ApoB100 protein contained in MDA-LDL is assumed to have undergone chemical modification involving MDA, or oxidation involving reactive oxygen species themselves.

At that time, it is assumed that the amino group (—NH ₂ ) on the side chain of the Lys residue is directly converted to MDA and converted to —NH—CH═CH—CHO. When oxidation of Lys residue by MDA further proceeds, the side chain amino group (—NH ₂ ) of Lys residue finally becomes dihydorxypyridine (DHP). Changes in the molecular weight of lysine due to these chemical modifications involving MDA are MDA + 54 and DHP + 134. FIG. 10 shows a structure when Lys is subjected to MDA and a structure when Lys is subjected to DHP.

Also, oxidation of Met residues is envisaged. If Met residue is subjected to are oxidized chemically modified, in its side chain -S- is, -SO -, - SO ₂ - are converted to, + 16, + 32 molecular weight change in results.

The selected “peaks (pI _av. , (M / z) _av )” are all ionic species having a charge number z = 1, that is, ionic species [M _peptide + H] ⁺ type peaks. The (m / z) _av is in the range of 400 <(m / z) _av <15000, that is, an ion derived from a _peptide fragment (pI, M _peptide ) in the range of 400 <M _peptide + 1 <15000. Assume that it is a peak of the species [M _peptide + H] ⁺ .

At least the amino acid sequence of the peptide fragment (pI _av. , M _peptide ) in the range of 400 <M _peptide + 1 <15000 has a range of 4 to 50 amino acid residues.

First, a group of peptide fragments having 50 or less amino acid residues that may be generated due to trypsin digestion of ApoB100 protein that has not undergone oxidation is predicted based on the amino acid sequence information of ApoB100 protein. Specifically, all peptide fragments having 50 or less amino acid residues that may be generated as a result of trypsin digestion in the Lys residue and Arg residue contained in the amino acid sequence of the ApoB100 protein. Predict and make a group of “theoretical predicted peptide fragments” derived from the ApoB100 protein. Based on the amino acid sequence of each peptide fragment included in the group of “theoretical predicted peptide fragments” derived from ApoB100 protein, its molecular weight M _peptide is calculated. The “theoretical predicted peptide fragment” derived from the ApoB100 protein is generated by performing trypsin digestion by selecting any two of the Lys residue and Arg residue contained in the amino acid sequence of the ApoB100 protein. A peptide fragment in which the number of amino acid residues in the partial amino acid sequence is 50 or less. Therefore, the C-terminal of the “theoretical predicted peptide fragment” is a Lys residue or an Arg residue. In the amino acid sequence having 50 or less amino acid residues, a Lys residue or an Arg residue is further added. Some are included.

Regarding a peptide fragment containing a Lys residue, which is included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein, one of the contained Lys residues has a molecular weight when MDA-modified (M _peptide +54) Calculate Further, for a peptide fragment containing a plurality of Lys residues, the molecular weight (M _peptide + 54 × n ₁ ) when n ₁ of the included Lys residues has undergone MDA modification is calculated.

Similarly, with respect to peptide fragments containing Lys residues included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein, the molecular weight (M) of one of the Lys residues included was subjected to DHP modification. _peptide +134). Further, for a peptide fragment containing a plurality of Lys residues, the molecular weight (M _peptide + 134 × n ₂ ) when n ₂ of the contained Lys residues have undergone DHP modification is calculated.

In addition, for peptide fragments containing a plurality of Lys residues included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein, n _{2 of the} Lys residues included are subjected to DHP modification, and are included. The molecular weight (M _peptide + 54 × n ₁ + 134 × n ₂ ) when n _{1 of} Lys residues to be subjected to MDA modification is calculated.

Regarding peptide fragments containing Met residues, which are included in the group of “theoretical predicted peptide fragments” derived from the ApoB100 protein, one of the Met residues included is oxidized and converted into a monoxide (Met (O)). Calculate the molecular weight (M _peptide +16) when converted. Furthermore, for peptide fragments containing a plurality of Met residues, the molecular weight (M _peptide + 16 × n) when n _{3 of the} Met residues contained were oxidized and converted to a monoxide (Met (O)). ₃ ) Calculate.

Regarding peptide fragments containing Met residues, which are included in the group of “theoretical predicted peptide fragments” derived from the ApoB100 protein, one of the Met residues included is oxidized and becomes a dioxide (Met (O ₂ )). The molecular weight (M _peptide +32) when converted is calculated. Further, for peptide fragments containing a plurality of Met residues, the molecular weight (M _peptide + 32 × n) when n ₄ of the included Met residues are oxidized and converted into a dioxide (Met (O ₂ )). ₄ ) Calculate.

In addition, with respect to peptide fragments containing a plurality of Met residues included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein, n _{4 of the} Met residues included are expressed as dioxide (Met (O ₂ )), And the molecular weight (M _peptide + 16 × n ₃ + 32 × n ₄ ) when n _{3 of the} Met residues contained therein are converted into a monoxide (Met (O)) is calculated.

Molecular weight of peptide fragment without oxidation modification (M _peptide ) included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein;
Molecular weight of peptide fragment in which Lys residue contained is modified with MDA or DHP (M _peptide + 54 × n ₁ + 134 × n ₂ );
The molecular weight of the peptide fragment in which the Met residues involved are converted to monoxide (Met (O)) or dioxide (Met (O ₂ )) (M _peptide + 16 × n ₃ + 32 × n ₄ );
From the calculated “Molecular Weight Predicted Value (M _cal. )” List, the “Molecular Weight Predicted Value (M _cal. )” Is actually in the range of 400 <M _cal. +1 <15000 Is selected, and a “predicted molecular weight (M _cal. )” List of candidate peptide fragments is created.

From the “predicted molecular weight (M _cal. )” List of such candidate peptide fragments, it is estimated from (m / z) _{av of} the selected “peak (pI _av. , (M / z) _av )”. The candidate of “predicted molecular weight (M _cal. )” _That matches the molecular weight M _obs. = (M / z) _av −1 and the measurement error range (± 0.3) is selected.

That is, with respect to the selected “peak (pI _av. , (M / z) _av )”, based on only the molecular weight similarity, identification of the candidate of “predicted molecular weight (M _cal. )”, It is possible to determine (assign) amino acid sequences of candidate peptide fragments.

  As described more specifically in the “embodiment” described below, the peptide fragments shown in Table 13 and Tables 14 to 17 that satisfy the criterion of “AUC” value of 0.65 or more according to the present invention are “ Selected as a “biomarker”.

(Embodiment)
In this embodiment, according to the search process of a group of oxidized peptide fragments that can be used as “diagnostic markers” shown in FIG. "Isoelectric point protein separation chip", "Electrophoresis / drying device", "Solution coating device", "Mass spectrometer", "Computer for analysis program", and the specific analysis process used for .

  In this embodiment, since the peptide fragment to be analyzed is collected by “fragmentation with trypsin”, a group of peptide fragments that can be used as a searched “diagnostic marker” are shown in Table 13, Table 14 to Table 17 As described above, the cleavage ends of the peptide fragments are specific for trypsin digestion.

  In this embodiment, it has been clarified that peptide fragments containing the modified amino acids described in Table 18-1 and Table 18-2 can be used as a group of oxidized peptide fragments that can be used as “diagnostic markers”. Yes. In this embodiment, since the peptide fragment to be analyzed is collected by “fragmentation with trypsin”, the peptide fragment containing the modified amino acids described in Table 18-1 and Table 18-2 is cleaved. The end is selected from those specific to trypsin digestion, for example, a group of oxidized peptide fragments described in Tables 14-17.

  Peptide fragments containing the modified amino acids described in Table 18-1 and Table 18-2 become oxidized peptide fragments that can be used as “diagnostic markers” even when fragmented using digestive enzymes other than trypsin. . The technical scope of the “cardiac disease diagnostic marker” according to the present invention includes all peptide fragments containing the modified amino acids described in Table 18-1 and Table 18-2, and is fragmented using digestive enzymes other than trypsin. And a group of peptide fragments containing the modified amino acids described in Table 18-1 and Table 18-2.

  In addition, in the search process for a group of oxidized peptide fragments that can be used as “diagnostic markers” shown in FIG. 1, the peptide fragments to be analyzed are of course collected in the form of “fragmentation with trypsin”. Even in a form in which the peptide fragment is fragmented using a digestive enzyme other than trypsin, the method is similarly effective when searching for the “cardiac disease diagnostic marker” according to the present invention.

  The following is a result of searching for a group of peptide fragments that can be used as “diagnostic markers” and a specific analysis process according to the search process for a group of oxidized peptide fragments that can be used as “diagnostic markers” shown in FIG. .

  First, the “isoelectric point protein separation chip”, “electrophoresis / drying device”, “solution application device”, “mass spectrometer”, and “analysis program computer” used for the search of this embodiment will be described below. .

<1> Isoelectric point protein separation chip FIG. 5 shows a specific configuration of the isoelectric point protein separation chip used in the embodiment. FIG. 5B shows the appearance of the isoelectric point protein separation chip. FIG. 5A shows the arrangement of the channels provided on the chip substrate, and the width W and the total length L of the channels. FIG. 5C schematically shows the cross-sectional shape of the chip substrate and the lid of the chip, and shows the depth D of the channel when the lid is mounted and the positions of the reservoirs at both ends of the channel. (D) of FIG. 5 shows the outline and size of the periodic arrangement of pillar structures (columnar bodies) formed on the bottom surface of the flow path, and (E) of FIG. 5 is formed on the bottom surface of the flow path. Another example of the pillar structure (columnar body) is shown. In the isoelectric point protein separation chip used in this embodiment, the size of the flow path is selected as a width W2 mm, a total length L35 mm, and a depth D10 μm. In the pillar structure (columnar body) formed on the bottom surface of the flow path, each pillar has a width W _pillar 3.75 μm, a length L _pillar 12.5 μm, and a height H _pillar 10 μm. Are arranged periodically at regular intervals so that the longitudinal direction thereof coincides with the longitudinal direction of the flow path. Linear polyacrylamide is coated on the inner wall surface (side wall and bottom surface) of the channel and the wall surface of the pillar structure (columnar body). The linear polyacrylamide coat keeps the flow path hydrophilic, and at the same time, suppresses electroosmotic flow during isoelectric focusing.

  In addition, although the concrete numerical value is described about the size of the flow path used in this embodiment, and the columnar structure (pillar structure) provided in this flow path, these numerical values are the implementation of this invention. 2 shows an example of a flow path of an isoelectric point protein separation chip that can be used for the above and a columnar structure (pillar structure) provided in the flow path, and is not limited to the structure of this specific example.

<2> Electrophoresis / Drying Device FIG. 6 shows a specific configuration of the electrophoresis / drying device used in the embodiment. An isoelectric point protein separation chip is installed on a chip base (not shown). A chip temperature is controlled through a chip base by a temperature control mechanism (not shown). The drying mechanism of the electrophoresis / drying apparatus shown in FIG. 6 is a freeze-drying mechanism, and includes a sealing glass lid for constructing a sealed tank, a decompression device (not shown), and a vacuum exhaust port. The electrodes are inserted into reservoirs at both ends of the flow path, and are electrically connected to a power source and a controller (not shown) by wiring (not shown).

<3> Solution coating device (not shown)
SuperSigmaCM-V2, a dispenser device made by MUSASHI ENGINEERING, is used. The matrix solution used in this embodiment is 20 μmol / μL sinnamic acid, 0.05% (v / v) TFA, 2% (v / v) formamide, 70% acetonitrile (boiling point 82 ° C.), and further as a peptide for mass correction. 900 fmol / μL Bradykinin fragment 1-7 (M = 757.3997), 200 fmol / μL ACTH fragment 18-39 (human) (M = 2465.1989), 200 fmol / μL Insulin B chain oxidized (bovine) (M = 34944.6513).

<4> Mass spectrometer (not shown)
Kratos AXIMA-CFR plus which is MALDI-TOF MS made by Shimadzu is used. When installing the protein separation chip in the mass spectrometer, a dedicated target plate is used. The dedicated target plate is processed into a target plate having a recess measured so that the above-mentioned isoelectric point protein separation chip can be set with reference to a standard target plate provided by Shimadzu. An isoelectric point protein separation chip coated with a matrix is set in the depression of the dedicated target plate and installed in the mass spectrometer main body. The laser beam scanning is divided into 71 sections from the end of the flow path to each section, and pulse / laser light irradiation is performed a certain number of times in each section, and the spectrum obtained by each pulse / laser light irradiation is integrated. It is set as the measurement result of mass spectrometry spectrum.

<5> Computer for analysis program (not shown)
Data analysis described later was performed using a computer for analysis program.

  Peak intensity standardization is performed for each measurement result of the mass spectrometry spectrum of each section. Standardization of peak intensity is based on the peak intensity of Bradykinin fragment 1-7 measured in each section using the peak intensity of Bradykinin fragment 1-7, which has been added to the matrix at a constant concentration as a mass correction peptide, as an internal standard. The relative value for is the standardized peak intensity.

≪Sample analysis process≫
Sample pretreatment (capture, fragmentation, recovery)
1-1. Procedure for immunoprecipitation / digestion / recovery of serum sample using anti-MDA-LDL antibody (Operation 1-1-I)
Invitrogen's magnetic beads DynaBeads (solutions and magnetic beads can be easily separated using a magnet) are used. Prepare 20 μL of magnetic beads DynaBeads in suspension for each serum sample.

(Operation 1-1-II)
Anti-MDA-LDL antibody (8 μg per serum sample, Sekisui Medical) is adsorbed on magnetic beads.

  After the anti-MDA-LDL antibody is adsorbed, 300 μL of serum sample and 900 μL of HEPES buffer (0.1% SDS) are added to 20 μL of the magnetic bead suspension and suspended. Do time.

  As an anti-MDA-LDL antibody to be used, an anti-human MDA-LDL monoclonal antibody created by immunizing a mouse with MDA-LDL according to the method described in Japanese Patent No. 315587: antibody No. 29225 (Sekisui Medical Co., Ltd.) ) Is preferred.

(Operation 1-1-III)
After the antigen-antibody reaction, the magnetic beads are washed once with HEPES buffer. The magnetic beads are then washed with PBS buffer. In addition, the magnetic beads are rewashed with 10-fold diluted PBS buffer.

(Operation 1-1-IV)
20 μL trypsin solution (72 ng / μL, 25 mM ammonium bicarbonate) is added to the antigen-antibody reaction-washed magnetic beads, and digestion reaction is performed while shaking at 1200 rpm for 16 hours at 37 ° C. Do. After the digestion reaction, the magnetic beads are separated from the digested peptide solution using a magnet, and the peptide solution is recovered. The collected peptide solution is dried using a centrifugal evaporator. The dried peptide sample is stored at −30 ° C. until isoelectric focusing with a chip.

Isoelectric focusing 1-2. Isoelectric focusing using isoelectric focusing protein chip (Operation 1-2-I)
66% (v / v) cIEF gel, 3% (v / v) Carrier ampholyte (pI 3-10), 500 fmol / μL angiotensin II (pI 6.74), 500 fmol / μL fibrinopeptide B (pI 4.0), If necessary, an aqueous solution to which 2% (v / v) fluorescent markers (pI 4, 5.5, 9) are added is prepared. This aqueous solution is used as a sample preparation solution for the chip. The standard peptides having known isoelectric points, angiotensin II (pI6, 74), and fibrinopeptide B (pI4.0) are used as an isoelectric point index.

(Operation 1-2 II)
One peptide sample that has been dried is dissolved in 8 μL of a sample preparation solution (hereinafter referred to as a sample solution). Further, using a vortex, the sample solution is stirred for 3 minutes and allowed to stand at ice temperature for 30 minutes.

(Operation 1-2-III)
Immediately prior to isoelectric focusing, the sample solution is stirred for 3 minutes using a vortex. Apply 1 μL of sample solution to the reservoir located at the end of the flow path of the isoelectric point protein separation chip. A sample solution is introduced from the reservoir into the channel.

(Operation 1-2 IV)
7 μL each of 100 mM phosphoric acid solution (about pH 2) and 40 mM sodium hydroxide solution (pH 12) as electrode solutions is applied to the acid side reservoir and the alkali side reservoir located at both ends of the flow path.

(Operation 1-2V)
An electrode is set in each reservoir located at both ends of the flow path, and voltages of 1.5 kV, 0 V, and 3.0 kV are applied for 60 s, 30 s, and 720 s, respectively.

(Operation 1-2 VI)
Immediately after electrophoresis, the chip is cooled to -30 ° C. The sample solution is frozen while maintaining the separated state.

(Operation 1-2 VII)
The lid of the isoelectric point protein separation chip is removed while keeping the frozen sample solution in the flow path.

(Operation 1-2-VIII)
By removing the lid, the chip whose upper end of the flow path is opened is placed in a sealed environment in a cooled state and evacuated. In a cooled state, the sample is freeze-dried by evacuation.

(Operation 1-2-IX)
After lyophilization, the fluorescence marker separation state in each channel is monitored using a fluorescence scanner or a fluorescence microscope.

MALDI-TOF-MS mass spectrometry 1-3. Mass spectrometry using MALDI-TOF-MS (Operation 1-3-I)
Prepare matrix solution. The matrix solution to be prepared is an aqueous solution of 20 μmol / μL sinnamic acid, 0.05% (v / v) TFA, 2% (v / v) formamide, 70% acetonitrile, and 900 fmol / μL as a peptide for mass correction. Bradykinin fragment 1-7 (M = 757.3997), 200 fmol / μL ACTH fragment 18-39 (human) (M = 2465.1986), 200 fmol / μL Insulin B chain oxidized (bovine) (M = 3494.6513).

(Operation 1-3-3-II)
After monitoring the separation state of the fluorescent marker in each channel, an isoelectric point protein separation chip on which the sample has been lyophilized is placed and fixed on a chip stage maintained at 80 ° C. A matrix solution is applied onto the flow path of the isoelectric point protein separation chip using a solution application apparatus. Adjust the pressure setting of the solution applicator and apply approximately 7.5 μL of matrix solution to each flow path of the isoelectric point protein separation chip.

(Operation 1-3-3-III)
The applied matrix solution is dried. After drying, the isoelectric point protein separation chip is stored at −30 ° C. until the isoelectric point protein separation chip is installed in the mass spectrometer using a dedicated target plate.

(Operation 1-3-3-IV)
The isoelectric point protein separation chip is fixed in the recess of the dedicated target holder, and the dedicated target holder is installed in the mass spectrometer body.

(Operation 1-3-3-V)
The flow path with a total length of L35 mm is divided into 0.5 mm intervals, and a total of 71 sections are sequentially irradiated from the acid side section to the alkali side section, and pulsed laser light irradiation is performed. taking measurement.

Analysis 1
In “Analysis 1”, a total of 33 samples, ie, a disease group (number of coronary artery lesion branches 3) 10 samples and a control (non-patient) group (coronary artery lesion branches 0) 23 samples, were measured for each sample channel segment. Using the measurement results of the mass spectrometry spectrum, “sample profiling” and “data analysis / marker identification” are performed according to the following procedures.

In the process of “data analysis / marker identification”, the peak intensity I (pI, [M _peptide + H] of the ion species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) measured in each sample Sm ⁺ ) _Sm needs to be compared quantitatively. Therefore, for each sample, “normalized peak intensity: I (pI, [M _peptide + H] ⁺ ) _relative-Sm ” is used to perform “normalization” processing, and “normalized peak intensity: I (pI, [ M _peptide + H] ⁺ ) _normal-Sm ”is calculated.

Furthermore, “normalized peak intensity: I (pI, [M” for the ion species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) measured in each sample Sm (Sm = 1 to 33). _peptide + H] ⁺ ) _normal-Sm ”, and in all sample groups consisting of a total of 33 samples,“ normalized peak intensity: I (pI, [M _peptide + H] ⁺ ) _normal-Sm ” The “peak intensity: I (pI, [M _peptide + H] ^{+ of} ionic species [M _peptide + H] ⁺ derived from _peptide fragment (pI, M _peptide ), measured in each sample by performing“ Z scoring ” ) " _Is calculated (I (pI, [M _peptide + H] ⁺ ) _Z-score-Sm ).

The procedure of the “normalization” process and the “Z score conversion” process is as follows.
(1) Logarithmic conversion (ln (I (pI, [M _peptide + H] ⁺ ) _{relative -Sm} )) of standardized peak intensity (I (pI, [M _peptide + H] ⁺ ) _relative-Sm ) is performed.
(2) “Log normalized peak intensity: ln of ionic species [M _peptide + H] ⁺ derived from _peptide fragment (pI, M _peptide ) extracted in the flow path of each sample Sm (Sm = 1 to 33) (I (pI, [M _peptide + H] ⁺ ) _{relative −Sm} ) ”average; ln (I (pI, [M _peptide + H] ⁺ ) _relative ) _av.-Sm = (1 / N _peak ) Σln (I ( pI, [M _peptide + H] ⁺ ) _{relative −Sm} ) and “standard deviation: σ (ln (I (pI, [M _peptide + H] ⁺ ) _relative )) _Sm ” are calculated with respect to the “average value”. “Average value: ln (I (pI, [M _peptide + H] ⁺ ) _relative ) _av.-Sm ” calculated for each sample Sm (Sm = 1 to 33) and “standard deviation: σ (ln (I ( pI, [M _peptide + H] ⁺ ) _relative )) _Sm ”, and [(ln (I (pI, [M _peptide + H] ⁺ ) _relative-Sm )” (PI, [M _peptide + H] ⁺ ) _relative-Sm ) −ln (I (pI, [M _peptide + H] ⁺ ) _relative ) _av.-Sm ) / σ (ln (I (pI, [M _peptide + H] ⁺ ) _Relative )) _Sm ] is calculated and set as “normalized peak intensity: I (pI, [M _peptide + H] ⁺ ) _normal-Sm ”.
(3) Calculated for each sample Sm (Sm = 1 to 33), the “normalized peak intensity: I (pI, M, _peptide + H) ⁺ derived from the peptide fragment (pI, M _peptide )” “M _peptide + H] ⁺ ) _normal-Sm ”, and a total of 33 samples, among all sample groups, the “average value among all sample groups”; I (pI, [M _peptide + H] ⁺ ) _{normal . -av = (1/33) ΣI (} pI, [M peptide + H] +) and _normal-Sm, for the "average value of the total sample group", "the standard deviation in all sample group: sigma (I ( pI, [M _peptide + H] ⁺ ) _normal ) ”. Then. “Normalized peak intensity: I (pI, [M _peptide + H] ⁺ ) calculated for each sample using the calculated“ average value in all sample groups ”and“ standard deviation in all sample groups ” For “ _normal-Sm ”, [I (pI, [M _peptide + H] ⁺ ) _normal-Sm ) −I (pI, [M _peptide + H] ⁺ ) _normal-av. ) / σ (I (pI, [M _peptide + H ] ⁺ ) _Nprmal )] is calculated and used as the Z value (I (pI, [M _peptide + H] ⁺ ) _Z-score-Sm ).

"Sample Profiling" and "Data Analysis / Marker Identification"
I. For all 33 samples in total, mass spectrometry spectra measured in each section are stored in the database in the flow path of each sample Sm (Sm = 1 to 33).

  II. For each sample Sm (Sm = 1 to 33) channel, the pI value in each section of the channel is estimated. Fluorescent markers showing known pI contained in the sample solution, as well as deliberately added isoelectric point index peptides: angiotensin II (pI 6.74) and fibrinopeptide B (pI 4.0), such as trypsin Calculate (estimate) pI values in each compartment using spot position information (pI, M) of peptide groups with known isoelectric points common to all sample solutions, such as known peptide fragments derived from autolysis To do.

III. In the flow path of each sample Sm (Sm = 1 to 33), a peptide fragment (pI, M _peptide ) produced by trypsin digestion from ApoB100 protein, which is included in MDA-LDL, forms a spot. Extract the peak of the derived ion species [M _peptide + H] ⁺ . Ion intensity data (I (m / z) _i ) of the mass spectrometry spectrum measured in each section i (i = 1 to 71) of the flow path and an estimated value (pI (i)) of the pI value in the section Based on the above, in accordance with the following procedures III-1 to III-3, the peak of the ion species [M _peptide + H] ⁺ derived from the peptide fragment (pI, M _peptide ) forming the spot is extracted.

III-1. Using the Savitzky-Golay filter, the ion intensity data (I (m / z) _i ) of the mass spectrometry spectrum measured in each section is m / z and ion intensity (I (m / z) By smoothing the mass spectrometry spectrum measured in each section by taking the average (moving average) of _i ). FIG. 7 shows an example of a mass spectrometry spectrum (I (m / z) _i-smoothing ) subjected to smoothing processing.

III-2. For each section i, the portion showing the maximum value is extracted as a “peak candidate” in the mass spectrometry spectrum (I (m / z) _i-smoothing ) of the section i subjected to the smoothing process. .

III-3. The ion intensity (I (m / z) _i-smoothing ) of the “candidate peak” extracted in the mass spectrometry spectrum measured in the adjacent section is expressed as m / z and pI value in each section i. Plotting on the two-dimensional coordinates (pI, m / z) of the estimated value (pI (i)) and connecting them, the final “peak” is extracted. In contrast to the “peak candidates” extracted in each section i, if there are “peak candidates” having substantially the same m / z in adjacent sections, these are expressed on two-dimensional coordinates (pI, m / z). “Peak candidates” are connected to form a “peak” indicating one common “peak position (m / z)” in the mass spectrometry spectrum. By this “peak” extraction operation, two-dimensional coordinates (pI, m / z) are obtained from a set of ion intensity data (I (m / z) _i ) of the mass spectrometry spectrum measured in each section in one channel. When plotted above, the “peak position (m / z)” is the same, and the “ion species derived from the peptide fragment (pI, M _peptide ) forming a spot that shows a spread in the pI direction [M _peptide + H ] ⁺ Peak "can be extracted. FIG. 8 shows an example in which “peaks” are extracted by concatenating “peak candidates”. In the extracted “peak”, the pI indicating the maximum of the ion intensity is searched in the pI direction, and is set as the pI value (pI _mean-Sm ) of the spot center of the “peak”. Further, the “peak position (m / z)” in the pI value (pI _mean-Sm ) of the spot center of the “peak” is set as the center position (m / z) _mean-Sm of the extracted “peak”. .

IV. Extracted from between different channels “Peak (pI _mean-Sm , (m / z) of ion species [M _peptide + H] ⁺ derived from _peptide fragments (pI, M _peptide ) forming spots” _"mean-Sm )" (signal matching). An allowable range of pI and m / z (isoelectric point direction ± 1, molecular weight direction ± 0.3) is set, and “peaks” existing within the ranges are regarded as the same “peak”. For a channel that does not have a corresponding “peak” within the allowable range, it is considered that there is no corresponding “peak” in the channel.

V. For all of the 33 samples in total, for the “peaks” associated with the different flow paths, the average value (pI _av. ) And (m / z) _{mean of the} central pI value (pI _mean-Sm ) _As the “peak” indicating the two-dimensional coordinates (pI _av. , (M / z) _av. ) _Consisting of the average value of _−Sm ((m / z) _av. ), The “standardized peak intensity: I (pI _{av .,} to create a list of _{(m / z) av) relative} -Sm ". In order to compare the peak intensities of the associated “peaks” between the flow paths of the samples Sm (Sm = 1 to 33), the above “normalization” process is performed on the “standardized peak intensities”. And “normalized peak intensity: I (pI _av. , (M / z) _av ) _normal-Sm ” is calculated. With respect to all 33 samples in total, the “normalized peak intensity: I () is defined as the“ peak ”indicating the two-dimensional coordinates (pI _av. , (M / z) _av. pI _av. , (m / z) _av ) _normal-Sm ”.

VI. For a group of “peaks” indicating the two-dimensional coordinates (pI _av. , (M / z) _av. ) _Associated with all 33 samples in total, the “normalized peak intensity: I (pI _{av .,} based on _{(m / z) av) normal} -Sm list of "disease group (coronary lesions branch number 3) 10 samples, control (non-patient) group (coronary lesions branch number 0) 23 two subgroups of the sample In the meantime, it is examined whether or not “normalized peak intensity: I (pI _av. , (M / z) _av ) _normal-Sm ” has a statistically significant difference. Specifically, a control (non-patient) group (number of coronary artery lesion branches 0) 23 is selected from a group of “peaks” indicating two-dimensional coordinates (pI _av. , (M / z) _av. ) 23 associated with each other. Compared with the sample, the “normalized peak intensity: I (pI _av. , (M / z) _av ) _normal-Sm ” is statistically significantly larger in 10 samples of the disease group (number of coronary artery lesion branches 3) _. “Peak” is selected. Alternatively, compared to 23 samples in the control (non-patient) group (0 coronary lesions), 10 samples in the disease group (3 coronary lesions) “normalized peak intensity: I (pI _av. , (M / z) _av ) _Select “peaks” where “ _normal-Sm ” is statistically significantly smaller.

With respect to a group of “peaks” indicating two-dimensional coordinates (pI _av. , (M / z) _av. ) _Associated with all 33 samples in total, the “normalized peak intensity: I (pI _av. , (M / z) _av ) _normal-Sm ”in order to more efficiently compare“ normalized peak intensity: I (pI _av. , (M / z) _av ) _normal-Sm ” “Z score” processing is performed, and “Z value: I (pI, [M _peptide + H] ⁺ ) _Z-score-Sm ” is calculated.

When the “Z value” is a positive value, the “normalized peak intensity: I (pI _av. , (M / z) _av ) _normal-Sm ” is “average value in all sample groups”; I ( pI _av. , (m / z) _av ) _normal-av. = (1/33) ΣI (pI _av. , (m / z) _av ) means greater than _normal-Sm . The average value of the calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” was calculated for 10 samples of the disease group (3 coronary lesions), and the disease group ( Number of coronary lesions 3) The average value of the calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” in a subgroup of 10 samples is at least 0.65 or more The “peaks” are selected.

The “peaks” selected based on the above criteria are “normalized peaks” in 10 samples in the disease group (3 coronary lesion branches) compared to 23 samples in the control (non-patient) group (0 coronary lesion branches). Intensity: I (pI _av. , (M / z) _av ) _normal-Sm ”corresponds to a statistically significant“ peak ”. Actually, since the total of the “Z value” for all 33 samples is 0, the calculated “Z value: I in the subgroup of the control (non-patient) group (coronary artery lesion branch number 0) 23 samples”. The average value of (pI _av. , (M / z) _av ) _Z-score-Sm is a more negative value than − (0.65) × (10/23) ≈−0.28. Yes. Therefore, the average value of the calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” in 10 samples of the disease group (coronary artery lesion branch number 3) and the control (non-patient) ) Group (0 coronary artery lesion branch number) in the sub-group of 23 samples, the difference between the calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” is It is at least 0.93 of “standard deviation in all sample groups: σ (I (pI _av. , (M / z) _av ) _normal )”, and may be regarded as having a statistically significant difference. Is possible.

Using the Z value (I (pI, M _peptide + 1) _Z-score-Sm ) of each peptide fragment (pI, M _peptide ) calculated for each individual Sm (Sm = 1 to 33), Perform receiver operating characteristic (ROC) analysis. The Z value (I (pI, M _peptide + 1) _Z-score-Sm ) of the peptide fragment (pI, M _peptide ) calculated from the measurement result of the sample derived from each individual Sm (Sm = 1 to 33) is large. Arranged in order, "ranking" from 1st to 33rd is made.

Disease group (coronary lesions branch number 3) 10 in a sample, Z value _{(I (pI, M peptide +1} ) Z-score-Sm) ratio is the number of samples is within the upper N ^th position (True positives rate or Sensitivity): TPR (pI, M _peptide +1: N ^th );
Control (non-patient) group (coronary lesions branch number 0) 23 in a sample, Z value _{(I (pI, M peptide +1} ) Z-score-Sm) ratio is the number of samples is within the upper N ^th position (False positives rate ): FPR (pI, M _peptide +1: N ^th );
Control (non-patient) group (coronary lesions branch number 0) 23 in a sample, Z value _{(I (pI, M peptide +1} ) Z-score-Sm) is the ratio of the number of samples not within the upper N ^th position (True negatives rate or Specificity), TNR (pI, M _peptide +1: N ^th );
The above three probabilities are calculated. Of course, FPR (pI, M _peptide + 1: N ^th ) + TNR (pI, M _peptide + 1: N ^th ) = 1.

Calculated on the ROC space, where the X-axis is TNR and the Y-axis is TPR (TNR (pI, M _peptide +1: N ^th ), TPR (pI, M _peptide +1: N ^th )) (N ^th = 1 ~ 33) are plotted. On the ROC space represented by (TNR, TPR), an ROC curve passing through (0, 1) and (1, 0) and the 33 plot points is drawn. The area A _plot of the region sandwiched between the drawn ROC curve and the X axis (Specificity) is calculated by numerical integration. The calculated area A _plot corresponds to AUC “area under the ROC curve”. Based on the criterion that the “AUC value” calculated by the ROC analysis is at least 0.60 or more, preferably 0.70 or more, a disease group (number of coronary artery lesion branches 3) and a control (non-patient) Peptide fragments (pI, M _peptide ) effective for estimating the group (number of coronary artery lesion branches 0) can be selected.

The total of 33 samples are composed of 10 samples of the disease group (number of coronary artery lesion branches 3) and 23 samples of the control (non-patient) group (number of coronary artery lesion branches 0), so that N ^th = 10 When the plot points (TNR (pI, M _peptide +1: 10), TPR (pI, M _peptide +1: 10)) match (1, 1) corresponding to perfect prediction in the ROC space, the “AUC value” is 1

Furthermore, among the 33 plot points (N ^th = 1 to 33), the “estimation criterion” adopted at the point closest to (1, 1) corresponding to perfect prediction in the ROC space is “ranking”. In the “estimation” method using “addition”, it can be regarded as an “estimation standard” that gives the “estimation” result with the highest accuracy.

Employed in "estimated" technique using "ranking" above threshold setting of "is within the upper N ^th position" is, Z value of the sample _{(I (pI, M peptide +1} ) Z-score _-Sm ) corresponds to setting a threshold value that is equal to or larger than the “Z value at the N ^th position (I (pI, M _peptide + 1) _Z-score-Sm )”. Therefore, the Z value (I (pI, M _peptide + 1) _Z-score-Sm ) of the sample is set to the threshold value Z value: I (pI, M _peptide + 1) _Z-score-Nth , I ( pI, M _peptide +1) _Z-score-Sm ≧ I (pI, M _peptide +1) Even if ROC analysis is performed for the “estimation” method using the “estimation criterion” that _Z-score-Nth , Essentially the same result.

Based on the selection criteria based on the “AUC value” calculated by the above ROC analysis, “peaks” whose “AUC value” is calculated to be at least 0.60 or more are selected. The amino acids of the _peptide fragment (pI, M _peptide ) produced by trypsin digestion from the ApoB100 protein included in MDA-LDL, which gives the “peak” in “Analysis 2” to be described later. The result of specifying the sequence is described.

Furthermore, the average value of the calculated “Z value: I (pI, M _peptide +1) _Z-score-Sm ” in 10 samples of the disease group (3 coronary lesions) is at least 0.65 or more. Based on the above criteria, the selected “peak” is also a peptide fragment (pI, M _peptide ) that is effective in estimating the disease group (number of coronary lesion branches 3) and the control (non-patient) group (number of coronary artery lesion branches 0). It corresponds.

The average value of the calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” in the subgroup of 10 samples of the disease group (3 coronary lesions) is at least 0 The “peak” selected based on the criterion of .65 or more is also generated by trypsin digestion from the ApoB100 protein contained in MDA-LDL that gives the “peak” in “Analysis 2” described later. The results of specifying the amino acid sequence of the peptide fragment (pI, M _peptide ) are described.

Actually, one of the “peaks” selected based on the selection criterion based on the “AUC value” and its two-dimensional coordinates (pI _av. , (M / z) _av. ) Are (pI _av. = 6.74) _. , (M / z) _av. = 1306) is calculated for 10 samples in the disease group (3 coronary lesions) compared to 23 samples in the control (non-patient) group (0 coronary lesions). FIG. 9 shows the results of verifying by t-test that “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” is statistically significant.

FIG. 9 shows the calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” for 23 samples of the control (non-patient) group (the number of coronary lesions is 0), The calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” is _divided and plotted in 10 samples of the disease group (number of coronary artery lesion branches 3). The distribution of the calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” for 23 samples in the control (non-patient) group (the number of coronary lesions is 0) and the disease group ( Number of coronary lesions 3) In 10 samples, there is a statistically significant difference from the calculated distribution of “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm ” When it was determined by t-test whether or not there was, it was determined that there was a significant difference at p = 0.007.

VII. The peptide fragment produced by trypsin digestion from ApoB100 protein contained in MDA-LDL, which gives the ionic species [M _peptide + H] ⁺ of (m / z) _av. The / MS method was applied to obtain internal sequence information regarding the amino acid sequence. From the result, the fragment is one of peptide fragments produced by trypsin digestion from ApoB100 protein contained in MDA-LDL. Its amino acid sequence is IISDYHQQFR (4487-4496, for amino acid residue numbers, Fragment (see ApoB100 protein amino acid sequence).

“Analysis 2”
In “Analysis 1”, the “peak” selected based on the selection criterion based on the “AUC value” is “the peptide fragment generated by trypsin digestion from ApoB100 protein contained in MDA-LDL” in the control ( Non-patient) group (number of coronary artery lesion branches 0) Compared with 23 samples, the generation rate is significantly higher in the disease group (number of coronary artery lesion branches 3) 10 samples, which is presumed to be more oxidized Judged to be.

In addition, in “Analysis 1”, the calculated “Z value: I (pI _av. , (M / z) _av ) _Z-score-Sm in a subgroup of 10 samples of the disease group (number of coronary artery lesion branches 3) "Peak" selected based on the criterion that the average value of "at least 0.65 or more" is also included in "peptide fragments produced by trypsin digestion from ApoB100 protein contained in MDA-LDL". Compared with 23 samples in the control (non-patient) group (0 coronary artery lesion branch), the generation ratio is significant in 10 samples of the disease group (3 coronary artery lesion branches), which are presumed to be more oxidized. It is judged to be high.

  In addition, in immunoprecipitation using an anti-MDA-LDL antibody, MDA-LDL is selectively fractionated, and the ApoB100 protein contained in the recovered MDA-LDL is chemically modified or activated by MDA. It is assumed that the oxygen species itself is undergoing oxidation.

At that time, it is assumed that the amino group (—NH ₂ ) on the side chain of the Lys residue is directly converted to MDA and converted to —NH—CH═CH—CHO. When oxidation of Lys residue by MDA further proceeds, the side chain amino group (—NH ₂ ) of Lys residue finally becomes dihydorxypyridine (DHP). Changes in the molecular weight of lysine due to these chemical modifications involving MDA are MDA + 54 and DHP + 134. FIG. 10 shows a structure when Lys is subjected to MDA and a structure when Lys is subjected to DHP.

Also, oxidation of Met residues is envisaged. If Met residue is subjected to are oxidized chemically modified, in its side chain -S- is, -SO -, - SO ₂ - are converted to, + 16, + 32 molecular weight change in results.

In “Analysis 2”, among the 10 samples of the disease group (3 coronary artery lesion branches), which are presumed to be more oxidized compared to 23 samples of the control (non-patient) group (0 coronary artery lesion branches). In all the “peaks (pI _av. , (M / z) _av )” selected in “Analysis 1” as “peaks” whose production ratio is significantly high, the “peaks” are given in “MDA-LDL” The peptide fragments produced by trypsin digestion from the ApoB100 protein contained in the above are identified.

The “peaks (pI _av. , (M / z) _av )” selected in “Analysis 1” are all ionic species having a charge number z = 1, that is, ionic species [M _peptide + H] ⁺ type peaks. Assume that there is. The (m / z) _av is in the range of 400 <(m / z) _av <15000, that is, it is derived from the peptide fragment (pI _av. , M _peptide ) in the range of 400 <M _peptide +1 <15000 _. It is assumed that it is a peak of the ion species [M _peptide + H] ⁺ .

At least the amino acid sequence of the peptide fragment (pI _av. , M _peptide ) in the range of 400 <M _peptide + 1 <15000 has a range of 4 to 50 amino acid residues.

First, a group of peptide fragments having 50 or less amino acid residues that may be generated due to trypsin digestion of ApoB100 protein that has not undergone oxidation is predicted based on the amino acid sequence information of ApoB100 protein. Specifically, all peptide fragments having 50 or less amino acid residues that may be generated as a result of trypsin digestion in the Lys residue and Arg residue contained in the amino acid sequence of the ApoB100 protein. Predict and make a group of “theoretical predicted peptide fragments” derived from the ApoB100 protein. Based on the amino acid sequence of each peptide fragment included in the group of “theoretical predicted peptide fragments” derived from ApoB100 protein, its molecular weight M _peptide is calculated. The “theoretical predicted peptide fragment” derived from the ApoB100 protein is generated by performing trypsin digestion by selecting any two of the Lys residue and Arg residue contained in the amino acid sequence of the ApoB100 protein. A peptide fragment in which the number of amino acid residues in the partial amino acid sequence is 50 or less. Therefore, the C-terminal of the “theoretical predicted peptide fragment” is a Lys residue or an Arg residue. In the amino acid sequence having 50 or less amino acid residues, a Lys residue or an Arg residue is further added. Some are included.

Regarding a peptide fragment containing a Lys residue, which is included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein, one of the contained Lys residues has a molecular weight when MDA-modified (M _peptide +54) Calculate Further, for a peptide fragment containing a plurality of Lys residues, the molecular weight (M _peptide + 54 × n ₁ ) when n ₁ of the included Lys residues has undergone MDA modification is calculated.

Similarly, with respect to peptide fragments containing Lys residues included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein, the molecular weight (M) of one of the Lys residues included was subjected to DHP modification. _peptide +134). Further, for a peptide fragment containing a plurality of Lys residues, the molecular weight (M _peptide + 134 × n ₂ ) when n ₂ of the contained Lys residues have undergone DHP modification is calculated.

In addition, for peptide fragments containing a plurality of Lys residues included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein, n _{2 of the} Lys residues included are subjected to DHP modification, and are included. The molecular weight (M _peptide + 54 × n ₁ + 134 × n ₂ ) when n _{1 of} Lys residues to be subjected to MDA modification is calculated.

Regarding peptide fragments containing Met residues, which are included in the group of “theoretical predicted peptide fragments” derived from the ApoB100 protein, one of the Met residues included is oxidized and converted into a monoxide (Met (O)). Calculate the molecular weight (M _peptide +16) when converted. Furthermore, for peptide fragments containing a plurality of Met residues, the molecular weight (M _peptide + 16 × n) when n _{3 of the} Met residues contained were oxidized and converted to a monoxide (Met (O)). ₃ ) Calculate.

Regarding peptide fragments containing Met residues, which are included in the group of “theoretical predicted peptide fragments” derived from the ApoB100 protein, one of the Met residues included is oxidized and becomes a dioxide (Met (O ₂ )). The molecular weight (M _peptide +32) when converted is calculated. Further, for peptide fragments containing a plurality of Met residues, the molecular weight (M _peptide + 32 × n) when n ₄ of the included Met residues are oxidized and converted into a dioxide (Met (O ₂ )). ₄ ) Calculate.

In addition, with respect to peptide fragments containing a plurality of Met residues included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein, n _{4 of the} Met residues included are expressed as dioxide (Met (O ₂ )), And the molecular weight (M _peptide + 16 × n ₃ + 32 × n ₄ ) when n _{3 of the} Met residues contained therein are converted into a monoxide (Met (O)) is calculated.

Molecular weight of peptide fragment without oxidation modification (M _peptide ) included in the group of “theoretical prediction peptide fragments” derived from the ApoB100 protein;
Molecular weight of peptide fragment in which Lys residue contained is modified with MDA or DHP (M _peptide + 54 × n ₁ + 134 × n ₂ );
The molecular weight of the peptide fragment in which the Met residues involved are converted to monoxide (Met (O)) or dioxide (Met (O ₂ )) (M _peptide + 16 × n ₃ + 32 × n ₄ );
From the calculated “Molecular Weight Predicted Value (M _cal. )” List, the “Molecular Weight Predicted Value (M _cal. )” Is actually in the range of 400 <M _cal. +1 <15000 Is selected, and a “predicted molecular weight (M _cal. )” List of candidate peptide fragments is created.

"Predicted value of the molecular weight (M _cal.)" Such candidate peptide fragments from the list, sorted by "Analysis 1" a "peak _{(pI av., (M /} z) av) " of (m / z) _av The candidate of “predicted value of molecular weight (M _cal. )” _That matches the molecular weight M _obs. = (M / z) _av −1 estimated from the above and the measurement error range (± 0.3) is selected.

Molecular weight M _obs. = (M / z) estimated from (m / z) _{av of} “peak (pI _av. , (M / z) _av )” selected in “Analysis 1” by the above selection operation _. ) The candidates of “predicted molecular weight (M _cal. )” _Selected for _av −1 are shown in Table 13 and Table 14 to Table 17 below.

Table 13 shows the ionic species [M _peptide + H] ⁺ derived from the “peptide fragment without oxidation modification” in the “peak (pI _av. , (M / z) _av )” selected in “Analysis 1” _. Those identified as peaks are summarized.

  In addition, about each peptide fragment described in the said Table 13, the isoelectric point pI and molecular weight Mw are shown to following Table 13-2.

Table 14 shows a peptide fragment in which “Lys residue contained in the peptide chain is MDA-modified in the“ peak (pI _av. , (M / z) _av ) ”selected in“ Analysis 1 ” _{. Are} identified as peaks of the ion species [M _peptide + H] ⁺ derived from "."

Table 15 summarizes what is identified as a peak of the ionic species [M _peptide + H] ⁺ derived from “a peptide fragment in which DHP modification is generated in the Lys residue contained in the peptide chain”.

Table 16 shows a peptide fragment in which MDA-modified modification occurs in the Cs-terminal Lys residue of the peptide chain in the “peak (pI _av. , (M / z) _av )” selected in “Analysis 1” _{. Are} identified as peaks of the ion species [M _peptide + H] ⁺ derived from "."

Table 17 shows “Peptide Fragments in which Met Residues Included in Peptide Chains Oxidatively Modified” in “Peak (pI _av. , (M / z) _av )” selected in “Analysis 1” _. What is identified as a peak of the ionic species [M _peptide + H] ⁺ derived from is summarized.

In Table 17, the oxidation modification of the Met residue contained in the peptide fragment “GISTSAASPAVGTVG M D M DEDDDFSK” of number 601 and the peptide fragment “HEQD M VNGI M LSVEK” of number 603 Determined to be monoxide (Met (O)).

On the other hand, in Table 17, it is determined that the oxidation modification of the Met residue contained in the peptide fragment “FNEFIQNELQEASQELQQIHQYI M ALR” of number 602 is a dioxide (Met (O ₂ )) whose molecular weight increase is +32. Is done.

In Tables 13 and 14 to 17, candidates for “predicted molecular weight (M _cal. )” Selected for each “peak” are determined based on the amino acid sequence of the candidate peptide and the presence in the ApoB100 protein. The position (amino acid residue number) is used for description. In addition, for each “peak”, the value of “AUC” used as a selection criterion at the time of selection in “Analysis 1” is also described.

In the “amino acid sequence of the candidate peptide” shown in Table 14, in the amino acid sequence of the ApoB100 protein,
4145, 2402, 4461, 1311, 3229, 1852, 117, 3404,
1291st, 1295th, 2825th, 2829th, 3531st, 3537th, 616th, 620th, 3221st, 3227th, 2426th, 2428th, 2002th, 2004th, 2403th, 2410th, 2418th 2425th, 3995th, 4021th, 4034th,
It can be seen that the Lys residue at the above position has undergone MDA modification.

In the “amino acid sequence of candidate peptide” shown in Table 15, in the amino acid sequence of ApoB100 protein,
539, 1034, 745, 616, 114, 157, 4261, 293, 3973, 2139, 4187, 2575, 732,
2369, 2371, 4274, 4278, 3944, 3946, 3673, 3682, 2418, 2425, 612, 613, 3537, 3547, 4478, 4485,
It can be seen that the Lys residue at the above position has undergone DHP modification.

In the “amino acid sequence of the candidate peptide” shown in Table 16, in the amino acid sequence of the ApoB100 protein,
557, 1724, 2002, 2208, 3221, 1344,
It can be seen that the Lys residue at the above position has undergone MDA modification.

In the “amino acid sequence of the candidate peptide” shown in Table 17, in the amino acid sequence of the ApoB100 protein,
4011th, 4013th, 4382th, 750th, 755th,
It can be seen that the Met residue at the above position has undergone oxidative modification.

  Therefore, the “amino acid sequences of candidate peptides” shown in Table 14 to Table 17 are the progress of MDA modification or DHP modification to the Lys residue at the amino acid residue position shown in Table 18-1, and When evaluating the progress of oxidative modification to the Met residue at the amino acid residue position shown in Table 18-2, it is judged that the peptide fragment is suitable as an “evaluation marker”.

Analysis 3
In “Analysis 2”, the estimated molecular weight M of “peak (pI _av. , (M / z) _av )” selected in “Analysis 1” is estimated based on (m / z) _av of the “peak”. _obs. = (m / z) _av −1, which matches the measurement error range (± 0.3), the candidate of “predicted molecular weight (M _cal. )” and the amino acid sequence of the candidate peptide are identified ing. The “peak (pI _av. , (M / z) _av )” described in Table 13 and Table 14 to Table 17 all have a value of “AUC” of at least 0.64 or more. It has become. That is, among the 33 samples used in “Analysis 1”, compared to 23 samples in the control (non-patient) group (number of coronary artery lesion branches 0), in the disease group (number of coronary artery lesion branches 3) in 10 samples, It is determined that the generation ratio is significantly high.

Further, a plurality of “peaks (pI _av. , (M / z) _av )” selected in “Analysis 1” are combined, and “normalized peak intensity: I (pI _av. (M / z) _av ) _normal-Sm "sum {ΣI (pI _av. , (M / z) _av ) _normal-Sm } is subjected to the above-mentioned" Z scoring "process, and a plurality of types of" peaks " “Z value: {ΣI (pI _av. , (M / z) _av )} _Z-score-Sm ” is calculated. With respect to the “Z value: {ΣI (pI _av. , (M / z) _av )} _Z-score-Sm ” for a combination of such multiple “peaks”, the control (non-patient) group (the number of coronary lesions is 0) ) When the distribution in the 23 samples and the distribution in the disease group (number of coronary artery lesion branches 3) in 10 samples are compared, a significant difference is found in the distribution.

For example, the peak of number 207 shown in Table 13: LYSILK (2758-2763), the peak of number 301 shown in Table 14, GASGTTGTYQEW K DK (4133-4147), the peak of number 451 shown in Table 15 : Consider the combination of ELL K DLS K EAQEVFK (4271-4285).

Peak Number 207: LYSILK of (2758-2763) the "normalized peak intensity", is denoted by I (207) _normal-Sm, the "average value of the total sample group" of the I (207) _normal-Sm , I (207) _normal-av. = (1/33) ΣI (207) _normal-Sm , and “standard deviation in all sample groups” with respect to “average value in all sample groups” is represented by σ (I ( 207) written as _normal ). Also, the “normalized peak intensity” I (207) _normal-Sm of the peak of the number 207 is calculated by _performing the above-described “Z scoring” process, and the “Z value” is calculated as I (207) _Z− Indicated as _score-Sm . Among the 33 samples in total, 23 samples of the control (non-patient) group (the number of coronary lesion branches 0) and 10 samples of the disease group (the number of coronary artery lesion branches 3) were _subjected to ROC using the I (207) _Z-score-Sm. The “AUC” value for the peak of number 207 described in Table 13 calculated by analysis is 0.673.

Peak Number 301: GASGTTGTYQEW K DK of (4133-4147) the "normalized peak intensity", I (301) is denoted by _normal-Sm, the I (301) an average value in the "total sample group of _normal-Sm the "a _{I (301) normal-av.} = (1/33) ΣI (301) normal-Sm, for the" average value of the total sample group ", the" standard deviation of the total sample group "sigma ( I (301) _normal ). Further, the “normalized peak intensity” I (301) _normal-Sm of the peak of number 301 is calculated by _performing the above “Z scoring” process, and the “Z value” is calculated as I (301) _Z− Indicated as _score-Sm . Among the 33 samples in total, 23 samples of the control (non-patient) group (number of coronary artery lesion branches 0) and 10 samples of the disease group (number of coronary artery lesion branches 3) were _subjected to ROC using the I (301) _Z-score-Sm. The “AUC” value for the peak number 301 described in Table 14 and calculated by analysis is 0.769.

Peak Number 451: ELL K DLS K EAQEVFK of (4271-4285) the "normalized peak intensity", is denoted by I (451) _normal-Sm, the I (451) of the _normal-Sm "in all sample groups “Average value” is I (451) _normal-av. = (1/33) ΣI (451) _normal-Sm and “standard deviation in all sample groups” with respect to “average value in all sample groups” Indicated as σ (I (451) _normal ). Also, the “normalized peak intensity” I (451) _normal-Sm of the peak of the number 451 is calculated by _performing the above-described “Z scoring” process, and the “Z value” is calculated as I (451) _Z− Indicated as _score-Sm . Among the 33 samples in total, 23 samples of the control (non-patient) group (number of coronary lesion branches 0) and 10 samples of the disease group (number of coronary artery lesion branches 3) were _subjected to ROC using the I (451) _Z-score-Sm. The “AUC” value for the peak of number 451 described in Table 15 and calculated by analysis is 0.727.

Using I (207) _Z-score-Sm , I (301) _Z-score-Sm , I (451) _Z-score-Sm calculated for each individual Sm (Sm = 1 to 33) , Perform receiver operating characteristic (ROC) analysis. I (207) _Z-score-Sm , I (301) _Z-score-Sm , and I (451) _Z-score-Sm calculated for each individual Sm (Sm = 1 to 33) are large. Arranged in order, "ranking" from 1st to 33rd is made.

Disease group (number coronary artery disease 3) 10 in a sample, Z values _{I (207) Z-score-} Sm is within the upper N ^th position, Z values _{I (301) Z-score-} Sm is within the top N ^th position in it, Z value _{I (451) Z-score-} Sm is within the upper N ^th position, one of its satisfying sample number proportion of (True positives rate or Sensitivity): TPR (207 + 301 + 451: N ^th );
Control (non-patient) group (coronary lesions branch number 0) 23 in a sample, Z values _{I (207) Z-score-} Sm is within the upper N ^th position, Z values _{I (301) Z-score-} Sm is higher is within N ^th position, Z values _{I (451) Z-score-} Sm> 0 is within the upper N ^th position, the ratio of the number of satisfying samples that either (False positive rate): FPR ( 207+ 301 + 451: N ^th );
Control (non-patient) group (coronary lesions branch number 0) 23 in a sample, Z values _{I (207) Z-score-} Sm is not within the upper N ^th position, Z values _{I (301) Z-score-} Sm is higher N Z value I (451) not within ^th position, _Z-score-S not within upper N ^th position, ratio of the number of samples satisfying these three conditions (true negative rate or specificity), TNR (207 + 301 + 451: N ^th );
The above three probabilities are calculated. Of course, FPR (207 + 301 + 451 : N th) + TNR: has a ^{(207 + 301 + 451 N th} ) = 1.

TNR the X axis, the Y-axis and TPR, on ROC space, is calculated (TNR (207 + 301 + 451 : N th), TPR (207 + 301 + 451: N th)) (N th = 1 ~ 33) are plotted. On the ROC space, draw an ROC curve that passes through the 33 plot points (0, 1) and (1, 0). The area A _plot of the region sandwiched between the drawn ROC curve and the X axis (Specificity) is calculated by numerical integration. The calculated area A _plot corresponds to AUC “area under the ROC curve”. The “AUC value” calculated by the ROC analysis is an “AUC” value related to “a combination of the peak of number 207, the peak of number 301, and the peak of number 451” and was found to be 0.858.

This result is obtained in the group consisting of the above 33 samples in total.
Based on “the combination of the peak of number 207, the peak of number 301, the peak of number 451”, for example, the condition of N ^th = 10, “Z value I (207) _Z-score-Sm is within the top ten, When the Z value I (301) _Z-score-Sm is within the top 10 and the Z value I (451) _Z-score-Sm > 0 is within the top 10, the condition is Based on the estimation criterion “estimate samples of subjects having 3 coronary lesions”, whether each sample is a sample of subjects having 3 coronary lesions or not is estimated with higher accuracy. It can be seen that

  Specifically, by adopting “a combination of a peak of number 207, a peak of number 301, and a peak of number 451”, it is possible to eliminate “incorrect estimation” due to “variation” in each “peak”, In the “combination” as a whole, it is verified that it is possible to estimate whether or not there are 10 samples of the disease group (number of coronary artery lesion branches: 3) with higher accuracy.

“Peak (pI _av. , (M / z) _av )” selected in “Analysis 1” is distributed in the control (non-patient) group (number of coronary artery lesion branches 0) in 23 samples and the disease group ( Number of coronary lesions 3) When comparing the distribution in 10 samples, a significant “distribution difference” is found. As exemplified above, by adopting “combination of multiple types of peaks”, it is possible to eliminate “incorrect estimation” caused by “variation” in individual “peaks”. When the distribution in the 23 samples of the control (non-patient) group (0 coronary artery lesion branch) and the distribution in the 10 samples of the disease group (3 coronary artery lesion branch) are compared, the “distribution difference” existing between the two subgroups Is more clarified. Therefore, it is used for the purpose of discriminating the difference in the degree of progression of the “pathological condition” between the two subgroups of the control (non-patient) group (the number of coronary artery lesion branches is 0) and the disease group (the number of coronary artery lesion branches is 3). It is shown that the use of “a combination of plural kinds of peaks” as exemplified above as “diagnostic marker” is more effective.

  For example, using the selected “diagnostic marker”, whether the subject Sm-test belongs to the disease group (number of coronary artery lesion branches 3) or the control (non-patient) group (number of coronary artery lesion branches 0) A procedure for performing the estimation will be described.

First, according to the method described above, “peak (pI _av. , (M / z) of a peptide fragment derived from the ApoB100 protein in MDA-LDL, which is collected from a blood sample collected from the subject Sm-test _. ) _Av ) ”,“ normalized peak intensity: I (pI _av. , (M / z) _av ) _{normal-Sm-test} ”is calculated.

Regarding “peak (pI _av. , (M / z) _av )” of “diagnostic marker”, “normal peak intensity threshold: I (pI _av. , (M / z) _av ) _normal-th ” The “normalized peak intensity: I (pI _av. , (M / z) _av ) _{normal-Sm-test} ” of the subject Sm-test is compared.

As a result of the comparison, when the determination condition: I (pI _av. , (M / z) _av ) _{normal-Sm-test} ≧ I (pI _av. , (M / z) _av ) _normal-th is satisfied, the subject Sm -test belongs to the disease group (coronary artery lesion branch number 3); if the determination condition is not satisfied, the subject Sm-test is assumed to belong to the control (non-patient) group (coronary artery lesion branch number 0). Do.

The “normal peak intensity threshold: I (pI _av. , (M / z) _av ) _normal-th ” for “peak (pI _av. , (M / z) _av )” used for the estimation operation is, for example, It is possible to set according to the following procedure.

Specifically, in “Analysis 1”, each individual Sm in 33 samples in total consisting of 23 samples of control (non-patient) group (number of coronary artery lesion branches 0) and 10 samples of disease group (number of coronary artery lesion branches 3). “Normalized peak intensity: I (pI _av. , (M / z)” of the “peak (pI _av. , (M / z) _av )” calculated for (Sm = 1 to 33), respectively _. ) _Av ) _Normal-Sm ”and applying the“ Cross-valodation ”method to“ normal peak intensity threshold for “peak (pI _av. , (M / z) _av )” : (PI _av. , (M / z) _av ) _normal-th ”can be optimized.

For example, by applying the method of “leave-one-out cross-validation”, the “normal peak intensity threshold value: I (for peak (pI _av. , (M / z) _av )” by the following procedure: pI _av. , (m / z) _av ) _normal-th ”can be optimized.

  From a total of 33 samples, one individual Sm (Sm = t; t = 1 to 33) is taken out and set as a “testing set”, and a group Sm (Sm ≠ t) of the remaining 32 individuals is designated as “training example ( training set) ". There are 33 types of one individual Sm (Sm = t; t = 1 to 33) as “testing set”, and “training set” Sm (Sm ≠ t; 33 types are also created for t = 1 to 33).

  When one individual Sm (Sm = t; t = 1 to 33) as a “testing set (testing set)” belongs to a disease group (number of coronary artery lesion branches 3), “training set” Sm ( Sm ≠ t; t = 1 to 33) is composed of 9 samples belonging to a disease group (3 coronary lesion branches) and 23 samples of a control (non-patient) group (0 coronary lesion branches). On the other hand, when one individual Sm (Sm = t; t = 1 to 33) as a “testing set (testing set)” belongs to the control (non-patient) group (the number of coronary artery lesion branches is 0), “training example ( training set) ”Sm (Sm ≠ t; t = 1 to 33) is composed of 10 samples in the disease group (3 coronary lesion branches) and 22 samples in the control (non-patient) group (0 coronary lesion branches). Is done.

With respect to the created “training set” Sm (Sm ≠ t; t = 1 to 33), the judgment condition: I (pI _av. , (M / z) _av ) _normal-Sm ≧ I (pI _av. , (m / z) _av ) If the _normal-th is satisfied, the subject Sm belongs to the disease group (number of coronary artery lesion branches: 3); if the determination condition is not satisfied, the subject Sm It is estimated that the patient belongs to the group (coronary artery lesion branch number 0).

The “training set” Sm (Sm ≠ t; t = 1 to 33) includes 9 samples belonging to the disease group (3 coronary artery lesion branches) and the control (non-patient) group (0 coronary artery lesion branches). ) If it consists of 23 samples,
Disease group (number of coronary artery lesion branches: 3) Ratio of the number of samples satisfying the above judgment condition among 9 samples (True positive rate or Sensitivity): TPR (Sm ≠ t: I (pI _av. , (M / z) _av ) _{normal -th} );
Ratio of the number of samples satisfying the above judgment condition (False positive rate) among 23 samples in the control (non-patient) group (the number of coronary lesions is 0): FPR (Sm ≠ t: I (pI _av. , (M / z) _{av Normal-th} );
Of the 23 samples in the control (non-patient) group (the number of coronary artery lesion branches is 0), the ratio of the number of samples not satisfying the above-mentioned criteria (True negative rate or Specificity), TNR (Sm ≠ t: I (pI _av. , (M / z) _av ) _normal-th );
The above three probabilities are calculated. Of course, FPR (Sm ≠ t: I (pI _av. , (M / z) _av ) _normal−th ) + TNR (Sm ≠ t: I (pI _av. , (M / z) _av ) _normal−th ) = 1 It has become.

The “training set” Sm (Sm ≠ t; t = 1 to 33) is divided into 10 samples in the disease group (3 coronary lesion branches) and the control (non-patient) group (0 coronary lesion branches). If it consists of 22 samples to which it belongs,
Proportion of the number of samples satisfying the above judgment condition (True positive rate or Sensitivity) in 10 samples of disease group (number of coronary lesions: 3): TPR (Sm ≠ t: I (pI _av. , (M / z) _av ) _{normal -th} );
Fraction positive rate in 22 samples of control (non-patient) group (0 coronary lesions): FPR (Sm ≠ t: I (pI _av. , (M / z) _{av Normal-th} );
Of 22 samples in control (non-patient) group (coronary artery lesion branch number 0), the ratio of the number of samples not satisfying the judgment condition (True negative rate or Specificity), TNR (Sm ≠ t: I (pI _av. , (M / z) _av ) _normal-th );
The above three probabilities are calculated. Of course, FPR (Sm ≠ t: I (pI _av. , (M / z) _av ) _normal−th ) + TNR (Sm ≠ t: I (pI _av. , (M / z) _av ) _normal−th ) = 1 It has become.

A total of 33 types of “training set” Sm (Sm ≠ t; t = 1 to 33) to be created are respectively calculated TPR (Sm ≠ t: I (pI _av. , (M / z ) _Av ) _normal-th ) and TNR (Sm ≠ t: I (pI _av. , (M / z) _av ) _normal-th ), a total of 33 combinations, with the X axis as TNR and the Y axis as TPR Plot on the ROC space. On the ROC space, (0, 1), (1, 0) and the 33 plot points (TNR (Sm ≠ t: I (pI _av. , (M / z) _av ) _normal-th ), TPR ( Sm ≠ t: An ROC curve passing through I (pI _av. , (M / z) _av ) _normal-th )) is drawn. The area A _plot of the region sandwiched between the drawn ROC curve and the X axis (Specificity) is calculated by numerical integration. The calculated area A _plot corresponds to AUC “area under the ROC curve”. The "AUC value" calculated by ROC analysis of the "peak _{(pI av, (m / z} ) av.) " In the "normal peak intensity to a threshold _{value: I (. PI av, (} m / z) av) Based on “ _normal-th ”, it is regarded as “AUC value”, AUC (I (pI _av. , (m / z) _av ) _normal-th ) for the above determination condition.

“Normalized peak intensity: I” of the “peak (pI _av. , (M / z) _av )” calculated for each individual Sm (Sm = 1 to 33) in a total of 33 samples _. When (pI _av. , (M / z) _av ) _normal-Sm ”are arranged in descending order,“ ranking ”from 1st to 33rd is performed. To position 1-position and 33 that have been _"ranking." "Normalized peak _{intensity: (. PI av, (m} / z) av) I normal-Sm " and, respectively, "peak (pI _av, (m / z) _av ) ”as“ normal peak intensity threshold: I (pI _av. , (m / z) _av ) _normal-th ”,“ AUC value ”for the above determination condition calculated by the ROC analysis, AUC (I (pI _av. , (M / z) _av ) _normal-th = I (pI _av. , (M / z) _av ) is obtained.

33 types of AUC (I (pI _av. , (M / z) _av ) _normal-th = I (pI _av. , (M / z) _av )) obtained are _expressed as “normal peak intensity threshold: I (pI _av. , (m / z) _av ) _normal-th ”, the horizontal axis is ranked according to the order of“ normalized peak intensity: I (pI _av. , (m / z) _av ) _normal-Sm ”. The vertical axis is plotted as AUC (I (pI _av. , (M / z) _av ) _normal-th = I (pI _av. , (M / z) _av ). In the vicinity, AUC (I (pI _av. , (M / z) _av ) _normal-th = I (pI _av. , (M / z) _av ) is maximized. AUC (I (pI _av. , (m / z) _av ) “normal peak intensity for“ peak (pI _av. , (m / z) _av ) ”giving _normal-th = I (pI _av. , (m / z) _av ) Threshold: I (pI _av. , (M / z) _av ) _normal-th ”is an optimized“ normal peak intensity threshold: I (pI _av. , (M / z) _av ) _{norm al-th} ".

  For example, based on “a combination of a peak of number 207, a peak of number 301, and a peak of number 451”, the subject Sm-test belongs to the disease group (coronary artery lesion branch number 3) or the control (non-patient) group (coronary artery) When estimating whether the lesion belongs to 0), the following determination conditions can be set.

“Normal peak intensity threshold: I (207) _normal-th ” for “No. 207 peak”, “Normal peak intensity threshold: I (301) _normal-th ”, “No. 451” for “No. 301 peak” “Threshold of normal peak intensity: I (451) _normal-th ” for “peak” is set. "Normal peak intensity threshold: I (207) _normal-th ", "Normal peak intensity threshold: I (301) _normal-th ", "Normal peak intensity threshold: I (451) _normal-th " “Normalized peak intensity: I (207) _{normal-Sm-test} ”, “Normalized peak intensity: I (301) _{normal-Sm-test} ”, “Normalized peak intensity: I ( 451) _{normal-Sm-test} ”is compared.

Result of the comparison, the determination condition _{"I (207) normal-Sm-} test ≧ I (207) normal-th, I (301) normal-Sm-test ≧ I (301) normal-th, I (451) normal- _{If Sm-test} ≧ I (451) _normal-th is satisfied ”, the subject Sm-test belongs to the disease group (number of coronary artery lesion branches 3); if the determination condition is not satisfied, the subject Sm-test The test is estimated to belong to the control (non-patient) group (the number of coronary artery lesion branches is 0).

"Normal peak intensity threshold: I (207) _normal-th ", "Normal peak intensity threshold: I (301) _normal-th ", "Normal peak intensity threshold: I (451) used for the estimation operation “ _Normal-th ” can be set, for example, according to the following procedure.

Specifically, in “Analysis 1”, each individual Sm in 33 samples in total consisting of 23 samples of control (non-patient) group (number of coronary artery lesion branches 0) and 10 samples of disease group (number of coronary artery lesion branches 3). “Normalized peak intensity: I (pI _av. , (M / z)” of the “peak (pI _av. , (M / z) _av )” calculated for (Sm = 1 to 33), respectively _. ) _Av ) _normal-Sm ”and applying the“ cross-validation ”method,“ normal peak intensity threshold: I (207) _normal-th ”,“ normal peak intensity threshold: I (301) It is possible to optimize “ _normal-th ” and “threshold of normal peak intensity: I (451) _normal-th ”.

For example, by applying the method of “leave-one-out cross-validation”, “normal peak intensity threshold: I (207) _normal-th ”, “normal peak intensity threshold: I ( 301) _normal-th ”and“ threshold of normal peak intensity: I (451) _normal-th ”can be optimized.

  From a total of 33 samples, one individual Sm (Sm = t; t = 1 to 33) is taken out and set as a “testing set”, and a group Sm (Sm ≠ t) of the remaining 32 individuals is designated as “training example ( training set) ". There are 33 types of one individual Sm (Sm = t; t = 1 to 33) as “testing set”, and “training set” Sm (Sm ≠ t; 33 types are also created for t = 1 to 33).

  When one individual Sm (Sm = t; t = 1 to 33) as a “testing set (testing set)” belongs to a disease group (number of coronary artery lesion branches 3), “training set” Sm ( Sm ≠ t; t = 1 to 33) is composed of 9 samples belonging to a disease group (3 coronary lesion branches) and 23 samples of a control (non-patient) group (0 coronary lesion branches). On the other hand, when one individual Sm (Sm = t; t = 1 to 33) as a “testing set (testing set)” belongs to the control (non-patient) group (the number of coronary artery lesion branches is 0), “training example ( training set) ”Sm (Sm ≠ t; t = 1 to 33) is composed of 10 samples in the disease group (3 coronary lesion branches) and 22 samples in the control (non-patient) group (0 coronary lesion branches). Is done.

With respect to the created “training set” Sm (Sm ≠ t; t = 1 to 33), the judgment condition “I (207) _normal-Sm ≧ I (207) _normal-th , I (301) _{When normal-Sm} ≧ I (301) _normal-th or I (451) _normal-Sm ≧ I (451) _normal-th is satisfied ”, the subject (sample) Sm is a disease group (number of coronary artery lesion branches If the determination condition is not satisfied, it is estimated that the subject Sm (sample) belongs to the control (non-patient) group (the number of coronary artery lesion branches is 0).

The “training set” Sm (Sm ≠ t; t = 1 to 33) includes 9 samples belonging to the disease group (3 coronary artery lesion branches) and the control (non-patient) group (0 coronary artery lesion branches). ) If it consists of 23 samples,
Disease group (number of coronary artery lesions: 3) Proportion of the number of samples satisfying the above judgment condition (True positive rate or Sensitivity) among 9 samples: TPR (Sm ≠ t: I (207) _normal-th , I (301) _{normal- th} , I (450) _normal-th );
Control (non-patient) group (number of coronary lesion lesions 0) among 23 samples, the ratio of the number of samples satisfying the determination condition (False positive rate): FPR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th );
Of the 23 samples in the control (non-patient) group (the number of coronary artery lesion branches is 0), the ratio of the number of samples not satisfying the judgment condition (True negative rate or Specificity), TNR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th );
The above three probabilities are calculated. Of course, FPR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) + TNR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = 1.

The “training set” Sm (Sm ≠ t; t = 1 to 33) is divided into 10 samples in the disease group (3 coronary lesion branches) and the control (non-patient) group (0 coronary lesion branches). If it consists of 22 samples to which it belongs,
Proportion of the number of samples satisfying the above judgment condition (True positive rate or Sensitivity) in 10 samples of disease group (number of coronary lesions: 3): TPR (Sm ≠ t: I (207) _normal-th , I (301) _{normal- th} , I (450) _normal-th );
Fraction positive rate in 22 samples of control (non-patient) group (0 coronary lesions): FPR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th );
Of 22 samples in the control (non-patient) group (coronary artery lesion branch number 0), the ratio of the number of samples not satisfying the determination condition (True negative rate or Specificity), TNR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) _normal-th );
The above three probabilities are calculated. Of course, FPR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) + TNR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = 1.

A total of 33 types of “training set” Sm (Sm ≠ t; t = 1 to 33) to be created are calculated TPR (Sm ≠ t: I (207) _normal-th , I (207 301) combination of _normal-th , I (450) _normal-th ) and TNR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ), total 33 Are plotted on the ROC space where the X axis is TNR and the Y axis is TPR. On the ROC space, (0, 1), (1, 0) and the 33 plot points (TNR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ), TPR (Sm ≠ t: I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) ROC curve is drawn. The area A _plot of the region sandwiched between (Specificity) is calculated by numerical integration, and the calculated area A _plot corresponds to AUC “area under the ROC curve.” “AUC calculated by the ROC analysis described above” the value _{"is, I (207) normal-th} , I (301) normal-th, I (450) based on a combination of _normal-th," AUC value for the determination condition _{", AUC (I (207) normal} -th , I (301) _normal-th , I (450) _normal-th ).

“Normalized peak intensity: I (207) _normal-Sm ” and “normalized peak intensity: I (301) calculated for each individual Sm (Sm = 1 to 33) in the total of 33 samples. _When “ _normal-Sm ” and “normalized peak intensity: I (405) _normal-Sm ” are arranged in descending order, “ranking” from 1st to 33rd is made, respectively. "Normalized peak intensity: I (207) _normal-Sm ", "Normalized peak intensity: I (301) _normal-Sm ", "Normalized peak intensity: I" (405) _normal-Sm ”,“ normal peak intensity threshold: I (207) _normal-th ”,“ normal peak intensity threshold: I (301) _normal-th ”,“ normal peak intensity threshold: I (451) _normal-th ”, a total of 33 × 33 × 33 threshold combinations (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I (301) _normal-Sm , I (450) _normal-Sm ) are created.

A determination condition based on a total of 33 × 33 × 33 combinations of threshold values (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) is calculated by the ROC analysis. “AUC value”, AUC ((I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I ( 301) _normal-Sm , I (450) _normal-Sm )).

A total of 33 × 33 × 33 types of AUCs ((I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I ( 301) _normal-Sm and I (450) _normal-Sm )) are arranged in the order of increasing values, "ranking" is performed from the first rank to the (33 * 33 * 33) rank. 1st place AUC ((I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I (301) _normal-Sm , I ( 450) _normal-Sm ))) to give a combination of thresholds (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I ( 301) _normal-Sm and I (450) _normal-Sm ) are regarded as optimized threshold combinations (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ). It is possible to deceive.

In practice, the "ranked" AUC ((I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I (301 ) _Normal-Sm , I (450) _normal-Sm )), it can be determined that “there is no difference in value” from the top 1 to the 27th.

Combination of threshold values (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I (301) _normal-Sm , I (450) _normal-Sm ), the following determination conditions “I (207) _normal-Sm ≧ I (207) _normal-th , I (301) _normal-Sm ≧ I (301) _normal-th , I (451) _normal-Sm ≧ I (451) when any of _normal-th is satisfied ”, the subject (sample) Sm belongs to the disease group (number of coronary artery lesion branches 3); When the determination condition is not satisfied, if the subject Sm (sample) belongs to the control (non-patient) group (the number of coronary artery lesion branches is 0), for each individual Sm (Sm = 1 to 33) in a total of 33 samples To estimate.

Out of a total of 33 samples according to the judgment conditions based on the above-mentioned first to 27th “combination of threshold values (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th )” The following probabilities are calculated using the estimation results for each individual Sm (Sm = 1 to 33).

From the estimation results using the judgment conditions based on each “threshold combination (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th )”,
Proportion of the number of samples satisfying the above judgment condition (True positive rate or Sensitivity) in 10 samples of disease group (number of coronary lesions 3): TPR (I (207) _normal-th , I (301) _normal-th , I ( 450) _normal-th );
Control (non-patient) group (coronary artery lesion branch number 0) of 23 samples, the ratio of the number of samples satisfying the judgment condition (False positive rate): FPR (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th );
Control (non-patient) group (number of coronary artery lesion branches 0) of 23 samples (true negative rate or specificity), TNR (I (207) _normal-th , I (301) _{normal -th} , I (450) _normal-th );
The above three probabilities are calculated. Of course, FPR (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) + TNR (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = 1.

For each “threshold combination (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th )” from the top 1 to the 27th, the calculated TNR (I ( 207) _normal-th , I (301) _normal-th , I (450) _normal-th ) and TPR (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) , Plot on the ROC space with the X axis as TNR and the Y axis as TPR. A total of 27 plot points (TNR (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ), TPR (I (207) _normal ) plotted on the ROC space _-th , I (301) _normal-th , I (450) _normal-th )) and (1, 1) on the ROC space D (I (207) _normal-th , I (301) _{normal- th} , I (450) _normal-th )). When the calculated distance D (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th )) to (1, 1) on the ROC space is arranged in ascending order, 1 "Ranking" is made up to the 27th place.

Usually, the "ranked" AUC ((I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I (301) _{normal -Sm} , I (450) _normal-Sm )), which gives the first value among "threshold combinations (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th )" but during the "ranking" distance _{D (I (207) normal-} th, I (301) normal-th, I (450) normal-th)), 1 -position of the distance D (I (207) _{normal- th} , I (301) _normal-th , I (450) _normal-th )).

Rarely, in "ranking" distance _{D (I (207) normal-} th, I (301) normal-th, I (450) normal-th)), 1 -position of the distance D (I (207) _{normal -th, I (301) normal-} th, I (450) normal-th)) gives the combination of the "threshold _{(I (207) normal-th} , I (301) normal-th, I (450) normal- _th ) "is ranked" AUC ((I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) = (I (207) _normal-Sm , I (301 ) _Normal-Sm , I (450) _normal-Sm )) gives the first value, “threshold combination (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) ". In this case, the first-ranked distance D (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th )) is ranked first. ) _Normal-th , I (301) _normal-th , I (450) _normal-th )), “threshold combinations (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) "can be regarded as an optimized combination of threshold values (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ).

That is, by applying the “leave-one-out cross-validation” method, the first-order “threshold combination (I (207) _normal-th , I (301) _normal-th , I ( 450) _normal-th ) ”actually corresponds to“ optimum threshold combination ”based on the estimation results for each individual Sm (Sm = 1 to 33) in a total of 33 samples. Is possible.

Furthermore, by applying the “leave-one-out cross-validation” method, the first-order “threshold combination (I (207) _normal-th , I (301) _normal-th , I ( 450) _normal-th ) ”and“ threshold combinations (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th ) ”ranked 2nd or lower. When it is extremely small, based on the estimation result for each individual Sm (Sm = 1 to 33) in a total of 33 samples, a more appropriate “threshold combination (I (207) _normal-th , I (301) _{normal -th} , I (450) _normal-th ) "can be selected.

It should be noted that optimization is performed for each of “peak of number 207”, “peak of number 301”, and “peak of number 451” and has been optimized “threshold of normal peak intensity: I (207) _{normal-th -opt.} ", optimized" normal peak intensity threshold: I (301) _{normal-th-opt.} ", optimized" normal peak intensity threshold: I (451) _{normal-th-opt.} " It is also possible to use this combination of optimized threshold values (I (207) _{normal-th-opt.} , I (301) _{normal-th-opt.} , I (451) _{normal-th-opt.} ). The combination of the optimized threshold values (I (207) _{normal-th-opt.} , I (301) _{normal-th-opt.} , I (451) _{normal-th-opt.} ) Is at least in the top 1 to 8 Are included in “threshold combinations (I (207) _normal-th , I (301) _normal-th , I (450) _normal-th )”.

  In the “diagnosis of the disease state” in heart disease, the essential change is an oxidative change of “ApoB100 protein contained in LDL”. Therefore, if it is a peptide fragment containing the “oxidation site” shown in Table 18-1 and Table 18-2 that clearly reflects the difference in “degree of oxidation of ApoB100 protein” revealed by the present invention, Table 14 -It is effective as a "diagnostic marker" of heart disease in the same manner as the peptide fragment having the amino acid sequence described in Table 17. That is, the “cardiac disease diagnostic marker” according to the present invention is not limited to peptide fragments having the amino acid sequences described in Tables 14 to 17.

  The “cardiac disease diagnostic marker” according to the present invention is a serum sample prepared from a blood sample as part of a “blood test” using a blood sample collected from a subject in “diagnosis of a disease state” in heart disease. It is a useful biomarker when evaluating the oxidative change of “ApoB100 protein contained in MDA-modified LDL” contained therein. Therefore, the present invention includes, as part of “blood test” using a blood sample collected from a subject, “ApoB100 protein contained in MDA-modified LDL” contained in a serum sample prepared from the blood sample. It is suitably used for applications in which the evaluation of the oxidation change of is efficiently advanced.

  In addition, in the “cardiac disease diagnosis marker” according to the present invention, it is possible to adopt the forms of the invention described in Supplementary Notes 1 to 15.

(Appendix 1)
A heart disease characterized in that it is a peptide derived from the amino acid sequence sites described in Table 18-1 and Table 18-2 in ApoB100 protein, and one or more amino acid residues thereof are oxidized. Diagnostic marker.

(Appendix 2)
A group of markers for diagnosing heart disease, comprising at least one peptide fragment derived from a peptide having a partial sequence of ApoB100 protein described in Table 13 above.

(Appendix 3)
A cardiac disease diagnostic marker group comprising at least one peptide fragment derived from a peptide having a partial sequence of ApoB100 protein described in Table 14 to Table 17 above.

(Appendix 4)
A cardiac disease diagnostic marker group, which is the peptide according to (Appendix 3), wherein one or a plurality of amino acid residues are oxidized.

(Appendix 5)
A method for diagnosing a heart disease, comprising the step of simultaneously detecting a quantitative change in components of the cardiac disease diagnosis marker group described in (Appendix 2) and (Appendix 3).

(Appendix 6)
(Appendix 1) to a method for detecting a cardiac disease diagnostic marker according to (Appendix 4),
A method for detecting a diagnostic marker for heart disease, comprising a step of immunoprecipitation.

(Appendix 7)
The method for detecting a heart disease diagnostic marker according to (Appendix 6), further comprising a protease treatment step.

(Appendix 8)
The method for detecting a cardiac disease diagnostic marker according to (Appendix 7), wherein the protease is trypsin.

(Appendix 9)
The cardiac disease diagnostic marker according to (Appendix 1) or (Appendix 4), wherein the oxidation is a chemical change caused by MDA conversion of an amino acid residue.

(Appendix 10)
The method for detecting a diagnostic marker for heart disease according to any one of (Appendix 6) to (Appendix 8), wherein the oxidation is a chemical change caused by MDA conversion of an amino acid residue.

(Appendix 11)
The cardiac disease diagnostic marker according to (Appendix 1) or (Appendix 4), wherein the oxidation is a chemical change caused by oxygen addition to an amino acid residue.

(Appendix 12)
The method for detecting a marker for diagnosing heart disease according to any one of (Appendix 6) to (Appendix 8), wherein the oxidative change is a chemical change caused by oxygen addition to an amino acid residue. .

(Appendix 13)
A detection system for a cardiac disease diagnosis marker, comprising at least an isoelectric point protein / peptide separation chip and a mass spectrometer.

(Appendix 14)
A heart disease diagnosis system comprising the heart disease diagnosis marker detection system according to (Appendix 13).

(Appendix 15)
A heart disease diagnosis method comprising using the heart disease diagnosis system according to (Appendix 14).

101 “Sample Pretreatment” Step 102 “Isoelectric Focusing” Step 103 “MALDI-TOF MS Mass Spectrometry” Step 104 “Sample Profiling” Step 105 “Data Analysis / Marker Identification” Step 201 “Serum Preparation” Step 202 “Immunization” “Sedimentation processing” step 203 “fragmentation” processing step 204 “fragmented peptide recovery” step 300 isoelectric point protein separation chip 301 columnar body 302 channel 303 substrate 304 lid 305a reservoir 305b reservoir 306 lid substrate portion 307 lid resin layer 400 Electrophoresis / drying device 401 Chip storage unit 402 Power supply for electrophoresis 404 Temperature control mechanism 405 Exhaust line / drying mechanism

Claims (13)

A method for evaluating the progress of oxidation in ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject,
It has the following steps (Step 1) to (Step 6),
(Process 1)
Applying MDA-LDL from a blood sample collected from a subject by applying an immunoprecipitation method using an anti-MDA-LDL monoclonal antibody;
(Process 2)
A step of allowing trypsin to act on the ApoB100 protein contained in the fractionated MDA-LDL to prepare a group of peptide fragments derived from the ApoB100 protein that have been digested with trypsin;
(Process 3)
A group of peptide fragments derived from the ApoB100 protein digested with trypsin by applying isoelectric focusing method is fractionated into predetermined isoelectric points according to the isoelectric point of each peptide fragment. Separating into a plurality of peptide fragment subgroups fractionated based on points;
(Process 4)
About the partial group of a plurality of peptide fragments fractionated based on the isoelectric point, the mass of the peptide fragment (M _peptide ) contained in the partial group of each peptide fragment is used as a mass analysis means, and MALDI-TOF The peptide fragment contained in each peptide fragment subgroup was measured based on the isoelectric point (pI) and mass (M _peptide ), and the peptide fragment (pI, M _peptide ), and measuring the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from each peptide fragment (pI, M _peptide );
(Process 5)
From a group of identified peptide fragments (pI, M _peptide ), from the Lys residue at the amino acid residue position shown in Table 1-1 below, and the Met residue at the amino acid residue position shown in Table 1-2 At least one oxidized peptide fragment corresponding to a peptide fragment derived from the ApoB100 protein, comprising at least one Lys residue or Met residue selected from the group, wherein the Lys residue or Met residue is oxidized. Selecting one process;
(Step 6)
Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the selected oxidized peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured , Evaluating the progress of oxidation in the ApoB100 protein contained in the fractionated MDA-LDL;
In step 6, the evaluation of the progress of oxidation is as follows:
(Sub-step 6-1)
Identified peptide fragments (pI, M _peptide) the total number of kinds of peptide fragments contained in the group of the N _{total-peptide,} are included in the group of identified peptide fragments (pI, M _peptide), the total number N _Total- Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the peptide fragment of the _peptide type, the peak intensity I (pI, [M _peptide + H] ⁺ ) average value, I (pI, [M _peptide + H] ⁺ ) _av calculating step;
(Sub-step 6-2)
The measured value of peak intensity I (pI, [M _peptide + H] ⁺ ) of ionic species [M _peptide + H] ⁺ derived from the selected oxidized peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured and calculated ApoB100 protein contained in the fractionated MDA-LDL by comparing the average value of the measured peak intensity I (pI, [M _peptide + H] ⁺ ), I (pI, [M _peptide + H] ⁺ ) _av In which a content ratio of oxidized ApoB100 protein containing the selected oxidized peptide fragment is estimated;
(Sub-step 6-3)
The higher the content ratio of the oxidized ApoB100 protein that contains the selected oxidized peptide fragment in the ApoB100 protein contained in the fractionated MDA-LDL, the more the ApoB100 protein contained in the fractionated MDA-LDL. The step of evaluating that the degree of progress of oxidation is high;
An evaluation method characterized in that the progress of oxidation is evaluated on the basis of the steps (sub-step 6-1) to (sub-step 6-3). A method for evaluating the progress of oxidation in ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject,
It has the following steps (Step 1) to (Step 6),
(Process 1)
Applying MDA-LDL from a blood sample collected from a subject by applying an immunoprecipitation method using an anti-MDA-LDL monoclonal antibody;
(Process 2)
A step of allowing trypsin to act on the ApoB100 protein contained in the fractionated MDA-LDL to prepare a group of peptide fragments derived from the ApoB100 protein that have been digested with trypsin;
(Process 3)
A group of peptide fragments derived from the ApoB100 protein digested with trypsin by applying isoelectric focusing method is fractionated into predetermined isoelectric points according to the isoelectric point of each peptide fragment. Separating into a plurality of peptide fragment subgroups fractionated based on points;
(Process 4)
About the partial group of a plurality of peptide fragments fractionated based on the isoelectric point, the mass of the peptide fragment (M _peptide ) contained in the partial group of each peptide fragment is used as a mass analysis means, and MALDI-TOF The peptide fragment contained in each peptide fragment subgroup was measured based on the isoelectric point (pI) and mass (M _peptide ), and the peptide fragment (pI, M _peptide ), and measuring the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from each peptide fragment (pI, M _peptide );
(Process 5)
Selecting at least one peptide fragment selected from the group consisting of peptide fragments containing the amino acid residues shown in Table 2 below, from a group of identified peptide fragments (pI, M _peptide );
(Step 6)
Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the selected peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured , Assessing the degree of oxidation in the ApoB100 protein contained in the MDA-LDL taken;
In step 6, the evaluation of the progress of oxidation is as follows:
(Sub-step 6-1)
Identified peptide fragments (pI, M _peptide) the total number of kinds of peptide fragments contained in the group of the N _{total-peptide,} are included in the group of identified peptide fragments (pI, M _peptide), the total number N _Total- Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the peptide fragment of the _peptide type, the peak intensity I (pI, [M _peptide + H] ⁺ ) average value, I (pI, [M _peptide + H] ⁺ ) _av calculating step;
(Sub-step 6-2)
Measured value of peak intensity I (pI, [M _peptide + H] ⁺ ) of ionic species [M _peptide + H] ⁺ derived from the selected peptide fragment, calculated as I (pI, [M _peptide + H] ⁺ ) _measured In the ApoB100 protein contained in the fractionated MDA-LDL, the average value of the peak intensity I (pI, [M _peptide + H] ⁺ ) and I (pI, [M _peptide + H] ⁺ ) _av are compared. Estimating the content of oxidized ApoB100 protein that produces the selected peptide fragment by trypsin digestion;
(Sub-step 6-3)
The higher the content ratio of the oxidized ApoB100 protein that is produced by trypsin digestion of the selected peptide fragment in the ApoB100 protein contained in the fractionated MDA-LDL, the more it is contained in the fractionated MDA-LDL. A step of evaluating that the degree of oxidation is high in the ApoB100 protein;
An evaluation method characterized in that the progress of oxidation is evaluated on the basis of the steps (sub-step 6-1) to (sub-step 6-3). A method for evaluating the progress of oxidation in ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject,
It has the following steps (Step 1) to (Step 6),
(Process 1)
Applying MDA-LDL from a blood sample collected from a subject by applying an immunoprecipitation method using an anti-MDA-LDL monoclonal antibody;
(Process 2)
A step of allowing trypsin to act on the ApoB100 protein contained in the fractionated MDA-LDL to prepare a group of peptide fragments derived from the ApoB100 protein that have been digested with trypsin;
(Process 3)
A group of peptide fragments derived from the ApoB100 protein digested with trypsin by applying isoelectric focusing method is fractionated into predetermined isoelectric points according to the isoelectric point of each peptide fragment. Separating into a plurality of peptide fragment subgroups fractionated based on points;
(Process 4)
About the partial group of a plurality of peptide fragments fractionated based on the isoelectric point, the mass of the peptide fragment (M _peptide ) contained in the partial group of each peptide fragment is used as a mass analysis means, and MALDI-TOF The peptide fragment contained in each peptide fragment subgroup was measured based on the isoelectric point (pI) and mass (M _peptide ), and the peptide fragment (pI, M _peptide ), and measuring the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from each peptide fragment (pI, M _peptide );
(Process 5)
From among a group of identified peptide fragments (pI, M _peptide ), a peptide fragment containing the MDA-modified Lys residue shown in Table 3 below, and a peptide containing the DHP-modified Lys residue shown in Table 4 inside At least a Lys residue or Met selected from the group consisting of a fragment, a peptide fragment containing the MDA-modified Lys residue shown in Table 5 at its C-terminus, and a peptide fragment containing an oxidized Met residue shown in Table 6 Selecting at least one oxidized peptide fragment corresponding to the peptide fragment whose residue has undergone oxidation;
(Step 6)
Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the selected oxidized peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured , Evaluating the progress of oxidation in the ApoB100 protein contained in the fractionated MDA-LDL;
In step 6, the evaluation of the progress of oxidation is as follows:
(Sub-step 6-1)
Identified peptide fragments (pI, M _peptide) the total number of kinds of peptide fragments contained in the group of the N _{total-peptide,} are included in the group of identified peptide fragments (pI, M _peptide), the total number N _Total- Based on the measured value of the peak intensity I (pI, [M _peptide + H] ⁺ ) of the ionic species [M _peptide + H] ⁺ derived from the peptide fragment of the _peptide type, the peak intensity I (pI, [M _peptide + H] ⁺ ) average value, I (pI, [M _peptide + H] ⁺ ) _av calculating step;
(Sub-step 6-2)
The measured value of peak intensity I (pI, [M _peptide + H] ⁺ ) of ionic species [M _peptide + H] ⁺ derived from the selected oxidized peptide fragment, I (pI, [M _peptide + H] ⁺ ) _measured and calculated ApoB100 protein contained in the fractionated MDA-LDL by comparing the average value of the measured peak intensity I (pI, [M _peptide + H] ⁺ ), I (pI, [M _peptide + H] ⁺ ) _av In which a content ratio of oxidized ApoB100 protein containing the selected oxidized peptide fragment is estimated;
(Sub-step 6-3)
The higher the content ratio of the oxidized ApoB100 protein that contains the selected oxidized peptide fragment in the ApoB100 protein contained in the fractionated MDA-LDL, the more the ApoB100 protein contained in the fractionated MDA-LDL. The step of evaluating that the degree of progress of oxidation is high;
An evaluation method characterized in that the progress of oxidation is evaluated based on the steps (sub-step 6-1) to (sub-step 6-3). In step 5, at least one of the oxidized peptide fragments selected is
The evaluation method according to claim 3, wherein the MDA-modified Lys residue shown in Table 3 is selected from a group of peptide fragments contained therein. The anti-MDA-LDL monoclonal antibody used for fractionation of MDA-LDL by immunoprecipitation in Step 1 is a monoclonal antibody specific for MDA-converted ApoB100 protein contained in MDA-LDL. The evaluation method according to any one of claims 1 to 4, wherein: Moreover, the fractionation of the peptide fragment to which the isoelectric focusing method is applied in the step 3 is carried out using an isoelectric focusing protein separation chip. Evaluation method described in 1. Oxidative stress in the process of generating MDA-LDL from LDL based on the evaluation results of the degree of oxidation of ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject A method for estimating the level of
Includes the following process A and process B (process A)
The evaluation of the progress of oxidation in ApoB100 protein contained in MDA-LDL fractionated from a blood sample collected from a subject, ApoB100 protein contained in MDA-LDL according to claim 1 A step of using a method for evaluating the degree of progress of oxidation in
(Process B)
In the process in which the MDA-LDL is produced from LDL based on the evaluation result of the degree of oxidation of ApoB100 protein contained in the MDA-LDL to be collected from the blood sample collected from the subject, Estimating the level of oxidative stress in an environment in which MDA-LDL production has progressed;
In Step B, the estimation of the level of oxidative stress is
Oxidation of the environment in which the production of MDA-LDL has progressed in the process of producing MDA-LDL from LDL the higher the degree of oxidation in ApoB100 protein contained in the fractionated MDA-LDL An estimation method characterized by estimating a high level of stress.
Oxidative stress in the process of generating MDA-LDL from LDL based on the evaluation results of the degree of oxidation of ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject A method for estimating the level of
Includes the following process A and process B (process A)
The evaluation of the progress of oxidation in the ApoB100 protein contained in the MDA-LDL collected from the blood sample collected from the subject is included in the MDA-LDL of the present invention according to claim 2 A step performed using a method for evaluating the degree of oxidation in ApoB100 protein;
(Process B)
In the process in which the MDA-LDL is produced from LDL based on the evaluation result of the degree of oxidation of ApoB100 protein contained in the MDA-LDL to be collected from the blood sample collected from the subject, Estimating the level of oxidative stress in an environment in which MDA-LDL production has progressed;
In Step B, the estimation of the level of oxidative stress is
Oxidation of the environment in which the production of MDA-LDL has progressed in the process of producing MDA-LDL from LDL the higher the degree of oxidation in ApoB100 protein contained in the fractionated MDA-LDL An estimation method characterized by estimating a high level of stress.
Oxidative stress in the process of generating MDA-LDL from LDL based on the evaluation results of the degree of oxidation of ApoB100 protein contained in MDA-LDL collected from a blood sample collected from a subject A method for estimating the level of
Includes the following process A and process B (process A)
Evaluation of the progress of oxidation in ApoB100 protein contained in MDA-LDL fractionated from a blood sample collected from a subject, ApoB100 protein contained in MDA-LDL according to claim 3 A step of using a method for evaluating the degree of progress of oxidation in
(Process B)
In the process in which the MDA-LDL is produced from LDL based on the evaluation result of the degree of oxidation of ApoB100 protein contained in the MDA-LDL to be collected from the blood sample collected from the subject, Estimating the level of oxidative stress in an environment in which MDA-LDL production has progressed;
In Step B, the estimation of the level of oxidative stress is
Oxidation of the environment in which the production of MDA-LDL has progressed in the process of producing MDA-LDL from LDL the higher the degree of oxidation in ApoB100 protein contained in the fractionated MDA-LDL An estimation method characterized by estimating a high level of stress. Prior to the diagnosis of heart disease, the progress of oxidation in the ApoB100 protein contained in the MDA-LDL sampled from the blood sample collected from the subject is evaluated for the purpose of grasping the condition of the subject. In this case, as a marker used for the evaluation, a method for selecting an oxidized peptide fragment derived from the ApoB100 protein, which is suitable for the evaluation of the progress of the oxidation,
As a marker used for the evaluation,
At least one Lys residue or Met residue selected from the group consisting of the Lys residue at the amino acid residue position shown in Table 7-1 below and the Met residue at the amino acid residue position shown in Table 7-2 Selecting at least one oxidized peptide fragment corresponding to a peptide fragment derived from ApoB100 protein containing a group, wherein the Lys residue or Met residue is oxidized
A method for selecting a marker. Prior to the diagnosis of heart disease, the progress of oxidation in the ApoB100 protein contained in the MDA-LDL sampled from the blood sample collected from the subject is evaluated for the purpose of grasping the condition of the subject. In this case, as a marker to be used for the evaluation, a method for selecting a peptide fragment derived from the ApoB100 protein suitable for the evaluation of the progress of the oxidation,
As a marker used for the evaluation,
Select at least one peptide fragment selected from the group consisting of peptide fragments containing amino acid residues shown in Table 8 below.
A method for selecting a marker. Prior to the diagnosis of heart disease, the progress of oxidation in the ApoB100 protein contained in the MDA-LDL sampled from the blood sample collected from the subject is evaluated for the purpose of grasping the condition of the subject. In this case, as a marker used for the evaluation, a method for selecting an oxidized peptide fragment derived from the ApoB100 protein, which is suitable for the evaluation of the progress of the oxidation,
As a marker used for the evaluation,
A peptide fragment containing the MDA-modified Lys residue shown in Table 9 below, a peptide fragment containing the DHP-modified Lys residue shown in Table 10 inside, and the MDA-modified Lys residue shown in Table 11 at the C-terminus An oxidized peptide fragment corresponding to a peptide fragment selected from the group consisting of peptide fragments containing oxidized Met residues shown in Table 12 and at least Lys residues or Met residues undergoing oxidation Select at least one
A method for selecting a marker. At least one of the selected oxidized peptide fragments is
The method for selecting a marker according to claim 12, wherein the MDA-modified Lys residue shown in Table 9 is selected from a group of peptide fragments contained therein.