JP4597741B2 - In vitro diagnostic system based on protein, partial protein and / or partial peptide, or profile thereof - Google Patents

Description

  The present invention relates to the field of diagnostic techniques using biomarkers for diagnosis of diseases, evaluation of drug effects, prognosis of diseases, and early diagnosis of diseases in health examinations in the field of in vitro diagnostics. The present invention also relates to an analysis technique for screening biomolecules and the like effective for drug development such as search for drug discovery targets in the drug development field. Furthermore, the present invention relates to a technical field for measuring differences in abundance and abundance ratio of proteins using mass spectrometry of proteins and peptides in biological samples at the basic research level, clinical research level, and pharmaceutical diagnostic field.

  As a means for discriminating the difference using a sample exhibiting a normal state and a state other than the normal state, a technique that has been generally used in an in vitro diagnostic agent is a conventional technique. The most common in-vitro diagnostic agents are those that perform diagnostic tests by analyzing components in the blood. In the prior art in this field, the presence of a single specific protein in blood or a so-called peptide having a molecular weight of 10,000 or less, or the activity in the case of an enzyme protein is measured, and a normal (healthy person) sample and a disease sample are measured. It has helped diagnosis with the obvious difference. That is, measure the amount or activity of single or multiple specific proteins or peptides in a certain number of healthy subjects and samples from disease patients in advance, determine the range of abnormal values and normal values, and evaluate the samples in the same way Measurement is performed and inspection evaluation is performed depending on whether the value belongs to an abnormal value range or a normal value range.

  As a specific measurement method, a specific primary antibody labeled with an enzyme that develops color when a sample is directly or diluted in advance and reacted with a substrate with a single or a plurality of specific proteins or peptides. Using a secondary antibody, the enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunoassay (CLIA), which measures the amount of color of the sample, is bound to the primary or secondary antibody. Radioimmunoassay (RIA) that measures using a radioisotope, and enzyme activity assay that measures the product by color development by directly giving a substrate when the protein is an enzyme. There is also a method for analyzing enzyme degradation products by high performance liquid chromatography (HPLC). These measurement targets are proteins that have not undergone degradation or the like (hereinafter referred to as intact proteins), and Aβ42, which is the only poorly soluble degradation product of amyloid beta protein in Alzheimer's disease, is the only test method for measuring degradation products. This is a test method (see Patent Document 1) for measuring poorly soluble Aβ42, which is considered to be one of the onset factors of Alzheimer's disease, in bone marrow fluid and blood. Here, protein degradation refers to structural changes in the protein accompanied by peptide fragment formation, which is mainly caused by intracellular processing and degradation due to the life of the protein. In addition to this, protein digestion degradation refers to peptide fragments that occur outside the cell. Defined as including proteolysis that occurs.

Although measurement using mass spectrometry has not yet been performed as an in vitro diagnostic agent in clinical practice, protein mass spectrometry is widely used as a differential analysis technique for proteins at home and abroad at the research level. Originally, two-dimensional electrophoresis has been widely used as a conventional protein expression analysis technique. However, since detection sensitivity of proteins and peptides having a molecular weight of 10,000 or less is not sufficient, recently, there is a method of fractionating a biological sample by column chromatography and analyzing the protein and peptide contained in the fraction by mass spectrometry. Further, there is a method of mass spectrometry using a protein chip as a pretreatment instead of column chromatography, and a method of mass spectrometry using magnetic beads as a pretreatment. In addition, for the purpose of analyzing intact proteins, a method of performing mass spectrometry using the above method after digestion with trypsin or the like has also been reported (see Patent Document 2). However, in any case, utilizing the properties of intact proteins, fractionation is performed as it is, or protein molecules that are specifically adsorbed are selected and analyzed by mass spectrometry.
JP 07-51096 A JP 2004-333274 A

  Diagnosis of discriminating the difference using a sample obtained from a living body exhibiting a normal state and a state other than normal using the above-mentioned conventional technique is to detect a protein or its partial peptide alone, There has been no method for comprehensively detecting the partial protein or the partial peptide, obtaining the presence pattern or profile of the protein and the partial peptide, and discriminating and diagnosing a state other than normal and non-normal. In addition, the conventional method cannot accurately determine the degree of progression of the pathological condition of a subject suffering from a disease. Furthermore, in the conventional method, sensitivity and specificity are in a trade-off relationship, and there is no diagnostic system in which both sensitivity and specificity are sufficiently high.

  The problem to be solved by the present invention is to determine a difference between a normal state and a state other than normal in a sample obtained from a living body that exhibits a normal state and a state other than normal. The above partial protein such as the digested degradation product protein and / or one or more partial peptides such as the digested degradation product peptide and the abundance or abundance ratio thereof are measured to obtain a protein / partial peptide profile, It is possible to evaluate and discriminate between normal and non-normal states of the living body. Furthermore, the problem to be solved by the present invention is to accurately determine the degree of progression of a disease state, and to provide a diagnostic system having both high sensitivity and specificity.

  The present inventor has intensively studied a method for accurately evaluating and discriminating normal and non-normal states of a living body so that a protein existing as an intact protein in a normal state causes a decrease in translational synthesis in a non-normal state. To produce partial peptides (partial proteins) as various digestion degradation product peptides (digestion degradation product proteins) or translation synthesis as partial proteins or partial peptides due to splicing abnormalities in protein translational synthesis, etc. In a disease state that is an example of a state other than normal, the present inventors have found that the type of protein that is digested, decomposed or translationally synthesized and the type and amount of a partial protein or partial peptide that are produced vary as the disease progresses. By measuring the types and amounts of these proteins, partial proteins and partial peptides in a biological sample obtained from a living body, the present inventors can accurately distinguish between a normal state and a state other than normal, It has been found that the degree of disease progression and the like can be evaluated in a state other than normal. Furthermore, the present inventors further used a protein and a partial peptide such as a digested degradation product protein and a digested degradation product peptide by using enzyme immunosorbent assay and mass spectrometry, so that a large number of proteins and digested proteins can be digested at once. The inventors succeeded in measuring partial proteins and partial peptides such as degradation product proteins and digestion degradation product peptides, and completed the present invention.

That is, the aspect of this invention is as follows.
[1] When present as an intact protein in a living body in a normal state but present as at least one partial protein or partial peptide of the intact protein in a living body in a state other than normal, the protein, the partial protein, and the partial peptide A method for evaluating and discriminating a normal state and a non-normal state of a living body, a disease state, or a degree of progression of a disease state using at least one as a marker, wherein the intact protein, partial protein and / or partial peptide in a biological sample A method for evaluating and discriminating a normal state and a non-normal state, a disease state, or a degree of progression of a living body, comprising measuring a type, abundance and / or abundance ratio and obtaining a protein / partial peptide profile.
[2] When present as an intact protein in a living body in a normal state but digested and decomposed into at least one digestion degradation product protein or peptide in a living body in a state other than normal, the protein, digestion degradation product protein, and digestion degradation product peptide A method for evaluating and discriminating a normal state and a non-normal state, a disease state, or a degree of progression of a living body using at least one of the above as a marker, wherein the intact protein, digested degradation product protein and / or Measure the type, abundance and / or abundance ratio of digestion degradation product peptides, and evaluate and discriminate the progress of normal and non-normal states, pathologies or pathologies, including obtaining protein / partial peptide profiles The method of [1].
[3] The method according to [1] or [2], wherein two or more kinds of intact proteins, partial proteins, partial peptides, digestion degradation product proteins and / or digestion degradation product peptides are used as markers.
[4] The method according to any one of [1] to [3], wherein the intact protein is a protein present in blood.
[5] The method according to any one of [2] to [4], wherein the partial protein or the partial peptide is a digestion degradation product by the action of an intact protein protease in a normal living body.

[6] The method according to any one of [1], [3] and [4], wherein the partial protein or partial peptide is a partial expression product of an intact protein in a normal state organism.
[7] The method according to any one of [1] to [6], wherein the state other than the normal state is inflammation, precancerous lesion, cancer or advanced cancer.
[8] The method according to any one of [1] to [7], wherein the normal state, chronic hepatitis, cirrhosis and liver cancer can be evaluated and discriminated.
[9] The method according to any one of [1] to [8], wherein the detection is performed by an enzyme or fluorescently labeled antibody method or mass spectrometry.
[10] When present as an intact protein in a living body in a normal state but present as at least one partial protein or partial peptide of the intact protein in a living body in a state other than normal, the protein, the partial protein, and the partial peptide An apparatus for evaluating and discriminating a normal state and a non-normal state of a living body, a disease state, or a degree of progression of a disease state using at least one as a marker, specifically for an intact protein, a partial protein and / or a partial peptide An apparatus including a capturing unit including a substance to be bound, a measuring unit, and an analyzing unit.

[11] When present as an intact protein in a living body in a normal state but digested and decomposed into at least one digestion degradation product protein or peptide in a living body in a state other than normal, the protein, the digestion degradation product protein, and the digestion degradation product An apparatus for evaluating and discriminating a normal state and a non-normal state, a disease state, or a degree of progression of a living body using at least one of peptides as a marker, comprising an intact protein, a digestion degradation product protein and / or a digestion degradation [10] The apparatus according to [10], including a capture unit, a measurement unit, and an analysis unit including a substance that specifically binds to the product peptide.
[12] The apparatus according to [10] or [11], wherein a protein / partial peptide profile is obtained by immunoprecipitation and mass spectrometry.
[13] The apparatus according to any one of [10] to [12], wherein the capture part is a carrier on which an antibody or aptamer that specifically binds to an intact protein, partial protein and / or partial peptide is immobilized.
[14] The apparatus according to any one of [10] to [13], wherein the measurement unit is a light detection means or a mass spectrometer.
[15] When present as an intact protein in a normal state organism, but present as at least one partial protein or partial peptide of the intact protein in a non-normal state organism, the protein, the partial protein, and the partial peptide A kit for evaluating and discriminating a normal state and a non-normal state of a living body, a disease state of a state other than normal, or a degree of progression using at least one as a marker, and at least an intact protein, partial protein and / or partial peptide A kit comprising a carrier on which a substance that specifically binds to a solid phase is immobilized.

[16] When present as an intact protein in a living body in a normal state but digested and decomposed into at least one digestive degradation product protein or peptide in a living body in a state other than normal, the protein, the digested degradation product protein, and the digested degradation product A kit for evaluating and discriminating a normal state and a non-normal state of a living body using at least one peptide as a marker, a disease state of a state other than normal, or a degree of progression, comprising at least an intact protein, a digestion degradation product protein, and [15] The kit according to [15], comprising a carrier on which a substance that specifically binds to a digested degradation product peptide is immobilized.
[17] The kit according to [15] or [16], wherein the substance that specifically binds to the intact protein, partial protein and / or partial peptide is an antibody or an aptamer.

  According to the present invention, the type and amount of at least one intact protein and / or at least one digestion degradation product protein or digestion degradation product peptide or the like of a partial protein or partial peptide in a biological sample derived from a subject are determined. By measuring, it is possible to evaluate and determine whether the subject is in a normal state or in a state other than normal. In addition, when a state other than normal is a disease, it is possible to evaluate and determine the severity of the disease, that is, the degree of progression of the disease state.

  The present invention also provides a diagnostic system that is both extremely accurate and specific. The present invention enables highly accurate diagnosis for the first time. In addition, the present invention enables early diagnosis of diseases. The target diseases of the present invention are diverse, and are highly useful in many drug effect determinations.

  For example, in Alzheimer's disease, as described above, Aβ42, which is a poorly soluble degradation product of amyloid beta protein, is an amyloid protein precursor (APP) that is an intact protein by secretase, a proteolytic enzyme that is activated in a disease-specific manner. ; Amyloid precurcer protein) is detected in bone marrow fluid and blood due to degradation. However, in the detection, there is no diagnostic accuracy that can accurately detect the onset timing of the disease in the early diagnosis. On the other hand, there is no means by which secretase activation can be monitored outside the body. According to the present invention, a highly accurate Alzheimer diagnostic system can be provided by monitoring a plurality of proteins degraded by secretase and digested degradation product peptides thereof.

In cancer, at the same time as the generation of cancer cells, even in the state where one or more cancer cells are present, the cancer cells are in an environment where they can be pushed away and proliferate. At that time, normal tissues adhere to each other by an extracellular matrix (ECM) to form a tissue structure, and cancer cells cannot proliferate unless they are destroyed. Therefore, it is thought that cancer cells proliferate while degrading proteins constituting extracellular matrix including type I collagen by matrix metalloproteinases (membrane type matrix metalloproteinase type I; MT1-MMP) existing in the cell membrane. . Also known is a membrane matrix metalloproteinase type II; MT2-MMP expressed by fibroblasts of cancer tissue. In particular, cancer cells with strong metastatic potential and cancer cells with strong malignant transformation and strong angiogenic activity are known to have high activity of matrix metalloproteinase. In MT2-MMP-deficient mice, angiogenesis and tumor growth are known. Suppression of performance has been reported. It is considered that the degradation state of the protein constituting the extracellular matrix degraded by the matrix metalloprotease and the peptides of digestion degradation products, as well as serum and plasma proteins present in the blood, are also degraded by these proteases. It is possible to provide a means for activating proteolytic enzymes in the affected area and detecting those degrading peptides in the blood, thereby realizing a highly accurate cancer diagnostic system.

  Furthermore, the present invention includes products from diagnostic systems in the field of in vitro diagnostics, diagnostic kits and related diagnostic services, medical institutions and laboratory diagnostic companies, contract analysis from individual patients, and public research institutions and pharmaceutical companies. It can be applied to contract research.

  The present invention exists as an intact protein in a normal state in a subject's living body, but when a partial protein or partial peptide of the intact protein is produced in a state other than normal, the intact protein, the partial protein and / or Whether the subject is in a normal state or a non-normal state by detecting the type and amount of the partial peptide and simultaneously detecting the variation in the type and amount of the intact protein, the partial protein, and the partial peptide It is a method for evaluating and discriminating the state of disease in a state other than normal, or the degree of disease progression when suffering from a disease. Peptides generally refer to those in which amino acids having a molecular weight of 10,000 or less are linked, and the number of amino acid residues is from about several to about 50 or less. In the present invention, a partial protein and / or partial peptide of an intact protein can be used as a biomarker. When a partial peptide is used, it is a peptide having a partial amino acid sequence of a part of the amino acid sequence of the intact protein. The term "partial protein" refers to a protein having a partial amino acid sequence which is part of the amino acid sequence of an intact protein and having a molecular weight exceeding 10,000. In the present invention, the partial protein and partial peptide of an intact protein refer to proteins and peptides having a partial amino acid sequence of the amino acid sequence of the intact protein, and the partial protein or peptide in the expression synthesis process by transcription / translation. In some cases, it is produced as a partial peptide, or after being synthesized as an intact protein, it is digested and degraded in vivo to produce a digested degradation product protein or a digested degradation product peptide. This is because protein synthesis and control mechanisms are deregulated when the living body is in a state other than normal. That is, the present invention discriminates whether a subject is in a normal state or a state other than normal using the expression synthesis and / or digestion degradation of the protein in the living body as an index, and a pathological state in a case where the subject is in a state other than normal It is also a method for evaluating and discriminating the degree of progression of the disease when suffering from the disease. Here, evaluation discrimination includes determination of whether the subject is normal or non-normal, determination of whether the subject is afflicted with a disease (diagnosis of the disease), and the subject is afflicted with a disease. The determination of the severity of the disease and the risk of suffering from the subject's disease.

  When the living body is in a state other than normal, such as suffering from a disease, at least one protein that is intact in the normal state is affected in the state other than normal and the partial protein or partial peptide is generated There is. In addition, the expression synthesis of the intact protein itself is affected, and the amount of intact protein produced may vary. For example, in a state other than normal, the protein expression system may be affected, and the amount of protein synthesis may vary so as to decrease or disappear. In protein expression, splicing may occur during the transcription process, and an intact partial protein or partial peptide may be expressed. Furthermore, a protein expressed and synthesized as an intact protein may be digested and degraded in vivo to produce at least one specific partial protein fragment or partial peptide fragment that is a digested degradation product. At this time, the type and amount of the protein that is affected by expression synthesis or undergoes digestive degradation varies depending on the type and degree of a state other than normal, for example, the type of disease and the degree of progression of the disease. The kind and amount of a protein fragment or peptide fragment that is a partial protein or partial peptide or digestion degradation product also vary depending on the type and degree of a state other than normal, for example, the type of disease and the degree of progression of the disease. That is, when a biological sample such as serum is collected from a subject and the type of protein, partial protein and partial peptide and the amount of each type in the sample are measured, the type of protein, partial protein and partial peptide and the respective types A characteristic protein / partial peptide profile of the sample containing information about the amount is obtained. Here, the protein / partial peptide profile refers to an intact protein, a protein indicated by the type and abundance of the partial protein and the partial peptide, a presence pattern of the partial protein and the partial peptide. When a partial protein or partial peptide is an intact protein degradation product, it is referred to as a protein / digestion degradation product peptide profile. By analyzing this protein / partial peptide profile, it is possible to know whether the subject is in a normal state, in a state other than normal, or how abnormal the subject is. In this respect, the disease state can be determined by the method of the present invention, and in the present invention, the protein, its partial protein and its partial peptide can be used as a disease state discrimination biomarker.

  In the present invention, at least one protein, partial protein or partial peptide can be used as a biomarker, but preferably two or more, more preferably three or more, four or more, five or more, six or more, 7 More than 8 types, 8 types, 9 types, or 10 types of proteins, partial proteins or partial peptides are used. In this case, a combination of different proteins may be used, or a combination of a certain protein and a partial peptide or a partial peptide of a protein other than the protein may be used. Only a plurality of intact proteins may be used as biomarkers. As described above, by using a plurality of proteins, partial proteins or partial peptides as biomarkers, it is possible to more accurately determine a state other than normal or normal, and to accurately determine a state other than normal, for example, a disease progress state. Can be judged.

  In the present invention, the biomarker may be quantified or the presence or absence may be determined by qualitative. When the same type of protein, partial protein or partial peptide is used as a biomarker, it is better to quantify each biomarker to obtain an accurate concentration and then obtain a protein / partial peptide profile to make an accurate judgment. If the types of biomarkers increase, the presence / absence of each biomarker can be qualitatively measured, and a protein / partial peptide profile relating to the presence / absence can be obtained to enable accurate determination.

  The intact protein herein refers to a protein synthesized in a normal state and present in the living body, and includes pre-processed proteins and processed proteins such as pre-proproteins and proproteins. Protein degradation refers to changes in the structure of the protein that accompany the processing that takes place inside the cell and the generation of peptide fragments, mainly degradation due to the lifetime of the protein. In addition to this, digestion and degradation of proteins are partial protein fragments and peptides that occur outside the cell Defined as including proteolysis to produce fragments.

  In the present invention, the normal state of a living body refers to a healthy state, and typically refers to a state that does not suffer from a disease. Having a disease in this case refers to a case where the disease can be identified clinically. On the other hand, a state other than normal includes not only a state suffering from a disease but also a state in which clinical symptoms have not been manifested but can be suffered from a disease in the future. For example, whether or not a patient is afflicted with cancer can be determined only after the presence of the cancer tissue is actually found by observing a microscopic change in the pathological tissue. However, in reality, in the progression of a series of cancers, inflammation occurs from the normal state, followed by precancerous lesions, followed by the development of cancer and further progression to advanced cancer. Continue to progress to serious diseases. In the present invention, as a disease progresses, one or more specific proteins are affected in terms of expression, or undergo digestive degradation to produce partial proteins and partial peptides such as digested degradation product proteins and digestion degradation product peptides. By obtaining a protein / partial peptide profile comprising the protein and one or more partial proteins or partial peptides thereof, it is possible to determine whether or not the subject is in a normal state. In addition, when a state other than normal is a state suffering from a disease, it is possible to evaluate and determine how much the disease has progressed. Furthermore, if the disease is not normal but the severity of the disease is low, it may return to a normal state in the future or may progress to a more serious disease. For example, if you have inflammation, like other diseases, inflammation may subside and return to normal in the future, but in the case of inflammation, it will develop into precancerous lesions, cancer, and advanced cancer. There are things to do. In such cases, inflammation can be said to be at high risk of progressing to cancer.

  According to the method of the present invention, the magnitude of the risk of suffering from a subject's disease can also be evaluated, which is useful for preventive medicine. Furthermore, when a patient suffering from a disease is treated with medication, the condition goes in the opposite direction and the protein / partial peptide profile varies accordingly. By measuring this, it is possible to evaluate and determine the dosing effect. For example, when a partial protein or partial peptide of a certain protein is produced in a certain disease, the presence of the partial protein or partial peptide reflects the state in which the living body suffers from the disease, and the absence of the partial protein or partial peptide is Reflects the state in which the organism is not afflicted with a disease. When a drug that is a therapeutic agent for a disease is administered to a living body suffering from the disease, the abundance of the partial protein or partial peptide in the living body is reduced or disappears as the disease is cured. Therefore, the partial protein or partial peptide can be a biomarker for a drug effect determination endpoint. The method of the present invention enables biomarker search for a drug effect determination endpoint. It is also possible to predict the prognosis of the disease. Furthermore, drug discovery target biomolecules can be screened by the method of the present invention. Many intact proteins in the body act as physiologically active proteins. When the living body is in a normal state, the physiological activity of the protein is controlled by controlling the amount of expression and synthesis, processing by an enzyme, activation by an enzyme, and the like. An intact protein having physiological activity may have a control site for controlling the activity and an active site for exerting the activity, and the activity of the intact protein as a whole is controlled by the state of the living body. However, when the living body is in a state other than normal, the expression of intact protein is affected, or when digested and decomposed to produce a partial protein or partial peptide, the partial protein or partial peptide itself has an active site. However, if it does not have a control site, it may not be subject to activity control depending on the state of the living body. In this case, the partial protein or the partial peptide acts regardless of the state of the living body, and as a result, the living body is induced to a state other than normal, for example, a disease state. In addition, the partial protein or partial peptide itself may have a control site. In this case, the partial protein or partial peptide receives control that should be originally received by the intact protein. Deviations from activity control. Also in this case, the living body is induced to a state other than normal, for example, a disease state due to the influence of the partial protein or partial peptide. In this way, when a partial protein or partial peptide is a causative substance of a non-normal state such as a disease, the non-normal state can be restored to a normal state by suppressing the activity of the partial protein or partial peptide. Can do. In the method of the present invention, a protein / partial peptide profile in a state other than normal can be obtained, and the partial protein or partial peptide causing a disease or the like is determined by analyzing the activity of the partial protein or partial peptide. be able to. That is, drug discovery target biomolecules can be screened by the method of the present invention.

  In addition, in the present invention, the normal state of the living body includes a state that is not healthy but is not healthy, such as a state having mild inflammation, a state receiving stress, and a pregnancy state. Even in such a state that cannot be treated as a disease, in the normal state, the expression of a protein that exists as an intact protein in the living body is affected, or the protein is digested and degraded, and its partial digestion degradation product protein, partial digestion Partial proteins and partial peptides such as degradation product peptides, partial expression product proteins, partial expression product peptides and the like may be generated, and the protein / partial peptide profile may vary.

  The state other than normal targeted by the present invention is not limited, and examples thereof include advanced cancer, cancer, precancerous lesion, and inflammatory state before presenting precancerous lesion. Here, the precancerous condition refers to a condition in which malignant neoplasm is considered to occur in most cases, and includes cases that are clinically or not observed by microscopic changes in pathological tissue. . Cancers include esophageal cancer, stomach cancer, colon cancer, hepatocellular carcinoma, pancreatic cancer, bile duct cancer, breast cancer, lung cancer, lung adenocarcinoma, kidney cancer, bladder cancer, prostate cancer, uterus Body cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, ovarian cancer, skin cancer, leukemia, myeloma, lymphoma, lymphosarcoma, etc. are included. This includes the initial state where this occurred. Furthermore, for example, gastric ulcer, gastritis, hepatitis, cirrhosis, pulmonary fibrosis and the like are included. Furthermore, not only a disease or a similar state, but also a state in which a living body is stressed, a state of being pregnant, and the like can be determined by the method of the present invention. Here, the stressed state refers to physical stressors such as stimulation caused by high temperature or low sound, stimulation caused by radiation or noise, chemical stressors such as oxygen deficiency / excess, drug damage, nutritional deficiencies, and invasion of pathogenic bacteria. This refers to a state in which the living body shows a modulation as a result of the application of some stimulus such as a biological stressor, human relationship trouble, mental pain, mental stressor such as anger / anxiety / hate / tension to the living body.

  In the present invention, a protein that is present in an intact state in a normal state is affected by expression in a state other than the normal state, and the production amount of the intact protein varies, or a partial protein or partial peptide of a protein that is present in an intact state The mechanism generated by is not limited.

  For example, when a state other than normal is reached, the transcription / translation system of the protein is affected, the synthesis of the protein is reduced or inhibited, and the amount of the intact protein synthesized is changed. In addition, splicing becomes abnormal in the transcription of an intact protein, and a partial protein or partial peptide of the protein is synthesized. That is, a partial protein or partial peptide having a partial amino acid sequence of the amino acid sequence of an intact protein is synthesized.

  Furthermore, the mechanism by which intact proteins are digested and degraded is not limited, but examples include activation and high expression of proteolytic enzymes mainly in a state other than normal. For example, in a cancer state or a precancerous state, metalloproteases such as matrix metalloprotease, elastase, cathepsin and the like are activated in cells. In the present invention, digestion and degradation of proteins by these proteases are included, but digestion and degradation by other proteases and digestion and degradation not by proteases are also included. In addition, there is not only one mechanism by which a protein present in an intact state in a normal state is digested and decomposed in a state other than normal, and a plurality of mechanisms may be involved. For example, if the type of protease to be activated and the degree of activation differ in the state of disease progression, the type of protein that undergoes digestion degradation and the site that undergoes digestion degradation of the protein also change, and the type and amount of digestion degradation products The ratio also changes. Therefore, the protein / digestion degradation product peptide profile varies depending on the type and progression of the disease.

  When an intact protein is digested and degraded by a protease to produce a digested degradation product protein or digested degradation product peptide, the amount and / or activity of the protease can be measured by the method of the present invention. Therefore, the method of the present invention is a method for evaluating and discriminating the normal state and non-normal state of a living body, a disease state, or the progress of a disease state by measuring the amount and / or activity of a protease that degrades proteins in the living body. Is also included.

  The target intact protein is not limited, and is a protein that exists in the living body as an intact protein in a normal state, and when the living body is in a state other than normal, synthesis is reduced or lost, or transcription / translation. Any protein that is spliced in the process and synthesized as a partial protein or partial peptide, or digested and degraded to yield a digested degradation product protein or digested degradation product peptide is included. Such proteins include, for example, body fluid such as serum of a subject in a normal state and sample samples such as serum of a subject in a state other than normal by two-dimensional electrophoresis combining isoelectric focusing and SDS-PAGE. When the protein migrated by electrophoresis is stained by an appropriate method and visualized as a spot, the protein is a spot protein that exists in a normal state and disappears or becomes thin in a state other than normal. Therefore, when two-dimensional electrophoresis is performed on a biological sample obtained from a subject in a normal state and a biological sample obtained from a diseased patient, the protein in a spot that disappears or becomes thinner in the latter can be selected. Two-dimensional electrophoresis can be performed by a known method, or can be performed using a commercially available electrophoresis apparatus. Further, instead of two-dimensional electrophoresis, for example, two-dimensional HPLC using two types of chromatography may be used. The two types of chromatography in this case may be selected from known chromatography such as ion exchange chromatography, reverse phase chromatography, gel filtration chromatography and the like. At this time, by using an automatic apparatus that automatically performs two-dimensional HPLC and automatically performs mass spectrometry for each fraction, a large number of samples can be examined quickly. Examples of proteins include serum and plasma proteins present in blood. Serum and plasma proteins present in blood refer to proteins that can be produced in vivo and secreted into blood. Proteins present in the blood can be digested and degraded by proteases activated in lesions when passing through lesions in living organisms affected by the disease. Specifically, for example, α1-antitrypsin, haptoglobin, bradykinin, complement C3f, apolipoprotein C1, fibrinogen α chain, fibrinopeptide A like, complement C4A, apolipoprotein A1, inter α trypsin inhibitor, α2 macro Globulin, gelsolin, heparin cofactor II and the like. As examples of proteins that can be used as biomarkers in the method of the present invention, amino acids represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 28 Examples include proteins consisting of sequences. Which intact protein the partial protein or partial peptide whose profile varies between the normal state and the non-normal state can be determined by comparing the amino acid sequences of the two. In some cases, sugar chains are added to these partial proteins and partial peptides. As examples of biomarkers in the method of the present invention, such as partial proteins and partial peptides of the above proteins, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27 , 29, and the protein consisting of the amino acid sequence. The present invention also includes these partial peptides. The present invention relates to one or several amino acids in the amino acid sequence represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 29. Includes peptides having amino acid sequences deleted, substituted, or added, and these peptides can also be used as biomarkers in the method of the present invention. Here, “one or several” means “one or three”, “one or two” or “one”.

  Depending on the disease, a sugar chain may be added to an intact protein, its partial protein or its partial peptide. The present invention also includes a method for evaluating and discriminating a normal state and a state other than the normal state using the addition of these sugar chains as an index, and the proteins, partial proteins, and partial peptides to which these sugar chains are added are also used as biomarkers. obtain.

  FIG. 1 shows the concept of the present invention. FIG. 1 shows examples of intact protein fluctuations in a normal state in various states other than normal. In FIG. 1, the cases other than the normal state are myocardial infarction, hypertension, and cancer, but there are various variations, and the present invention is not limited to the example of FIG. For example, in a certain disease D1 (FIG. 1 left), protein X, which is present as an intact protein in a normal state, is digested and decomposed to produce peptides A, B and C. In another disease D2 (FIG. 1 middle), protein X undergoes digestion and degradation at a site different from that of D1, and peptides D and E are produced. Typically, protein X is digested and decomposed by a protease, the protease activated in disease D1 and disease D2 is different, and the site to be digested and decomposed is different. Although FIG. 1 shows an example in which a protein undergoes digestive degradation, the transcription and translation mechanism of protein A is affected in disease D1, and peptides A, B and C may be expressed and synthesized. In yet another disease D3 (FIG. 1 right), another protein Y that exists as an intact protein in the normal state is digested and decomposed to produce peptides F, G, and H. In the figure, the results of molecular weight measurement by intact mass, digested peptide, and peptide mass spectrometer are shown. Although not shown in the figure, the amount of intact protein also decreases or disappears due to digestion degradation. The type and amount of intact protein and each peptide present in each disease is a protein / partial peptide profile in each disease, and obtaining the profile indicates whether the subject is in a normal state or a state other than normal It is possible to know, and it is possible to know the state other than the normal state. That is, by previously associating each state with a protein / partial peptide profile, the state can be evaluated and discriminated from the obtained profile. Of course, it is not always necessary to obtain a profile for all of the intact proteins, each peptide, and by knowing one or more of these variations, the subject is in a normal state or in a state other than normal. In addition, it is possible to know the state other than the normal state. In addition, when obtaining a protein / partial peptide profile, a specific intact protein may be targeted, and the protein present in a biological sample and its partial peptide profile may be comprehensively obtained without limiting the protein. Good. By obtaining an exhaustive profile in this way, it is possible to determine any non-normal state related to the variation of the partial peptide from the intact protein.

  FIG. 2 shows the mechanism by which a protein is digested and decomposed to produce a partial peptide in a state other than normal. FIG. 2 shows the case of cancer as an example. FIG. 2 is also an example of the mechanism, and is not limited to this mechanism. In the case of FIG. 2, a protein activated in a cancer cell and having an increased protease activity in a blood vessel around the cancer cell and present in an intact state in a normal state is also an intact protein in the cancer cell. Although it is synthesized, it is digested and decomposed by the action of proteases in the blood vessels around the cancer cells when secreted from the cells into the blood, and the digested and decomposed peptides are present in the blood. The right side of FIG. 2 shows the protein / partial peptide profile in the normal state (top) and the protein / partial peptide profile in the state other than normal such as cancer (bottom). As shown in the right of FIG. 2, in the normal state, there are many intact proteins (arrows in the upper right of FIG. 2) in the blood, but in a state other than normal, the intact proteins are digested and decomposed into partial peptides. As a result, the amount of intact protein (arrow at the lower right in FIG. 2) decreases and the amount of the partial peptide having a smaller molecular weight (the peak at the left side of the arrow at the lower right in FIG. 2). Will increase. Although FIG. 2 shows a case where an intact protein is synthesized in a cancer cell, the synthesis site of the intact protein is not limited. For example, when an intact protein synthesized in a living tissue away from a cancerous part is secreted into the blood, the protease activity increases in that part of the blood vessel when passing through the blood vessel around the cancer cell. And may undergo digestive degradation.

  FIG. 3 shows a mechanism in which a protein is partially expressed and a partial peptide is generated in a state other than normal. The rectangular part in the upper left of FIG. 3 shows a cell. Proteins are synthesized by intracellular ribosomes, but in the normal state, intact proteins are synthesized and secreted as intact proteins (right in the upper left rectangle in FIG. 3). On the other hand, in a state other than normal, intracellular proteases are activated, intact proteins undergo digestion and degradation, and are secreted as partial peptides. Therefore, the protein / partial peptide profile in the blood in the normal state is as shown on the right side of FIG. 3, and the protein / partial peptide profile in the blood in the state other than the normal state is as shown in the lower side of FIG. The arrow in the figure showing the profile and the meaning of the peak are the same as in FIG.

  In the method of the present invention, a protein / partial peptide profile in a biological sample is examined, but the biological sample to be used is not limited. For example, liquid samples that can be collected from a living body, such as whole blood, serum, plasma, urine, saliva and the like can be mentioned. Depending on the storage state of the biological sample, the protein, partial protein and partial peptide in the biological sample may be further decomposed, and therefore it is preferable to use the biological sample without repeated freezing and thawing. Moreover, you may add a protease inhibitor etc. in a biological sample.

  The type and amount of protein in the biological sample can be measured by various methods. Although illustrated below, it is not limited to them. In the following example, an example of an intact protein and its partial peptide is shown, but a partial protein may be used instead of the partial peptide.

1. Enzyme-linked immunosorbent assay (ELISA)
An antibody against a specific intact protein and its partial peptide is bound to a carrier such as a microtiter plate which has been subjected to special chemical modification in advance. The bonding can be performed by a known method such as physical adsorption or covalent bonding using a functional group. The antibodies to be used are (1) an antibody that recognizes only the full length of a specific protein, (2) an antibody that recognizes only a partial peptide, (3) an antibody that recognizes both a specific protein and its partial peptide, or A combination of (1) and (2), (1) and (3), or (2) and (3) may be used. A sample is serially diluted with a stock solution or a buffer solution, and an appropriate amount thereof is added to a microtiter plate to which an antibody is bound, and incubated. Thereafter, washing is performed to remove uncaptured proteins and partial peptides. Next, a secondary antibody conjugated with a fluorescent or chemiluminescent substance or enzyme is added and incubated. For detection, evaluation is performed by adding each substrate and then measuring visible light by fluorescent or chemiluminescent substances or enzymatic reactions.

  Moreover, when there are more partial peptides, the types of antibodies to be used may be increased or the number of combinations may be increased.

  Although this example is a method using an antibody, a substance capable of binding to an intact protein or a partial peptide thereof may be used instead of the antibody. For example, an aptamer or the like can be used.

  In the present invention, the enzyme immunosorbent assay is present as an intact protein in a normal living body, but is digested and decomposed into at least one partial peptide produced in a living body other than normal, for example, a digestion-degrading peptide. For proteins, it also includes a device for evaluating and discriminating normal and non-normal states of a living body, including detecting the type and abundance or abundance ratio of the intact protein and its partial peptide in a biological sample. . The apparatus includes at least an antibody or aptamer immobilization part (capture part) and a measurement part. The antibody or aptamer immobilization part is composed of a solid phase carrier such as a slide glass or a 96-well titer plate on which the antibody or aptamer is immobilized. The measurement unit is composed of light detection means such as a spectrophotometer and a fluorescence spectrophotometer. Furthermore, the apparatus of the present invention may include an analysis unit that analyzes the obtained data, and the analysis unit includes a data processing device and analysis software.

2. Method using microarray (microchip) A microarray is a generic term for devices in which substances that can be bound to a substance to be measured are aligned (array) immobilized on a carrier (substrate). In the case of the present invention, as described in 1 above, antibodies or aptamers against intact proteins and partial peptides may be used after being aligned and immobilized. As the microarray carrier, various materials such as a slide glass and a nitrocellulose membrane can be used. Immobilization can be performed in the same manner as in the above case 1, but can be easily performed by using a commercially available microarrayer or spotter. In the microarray method, the amount of the immobilized phase is small, and the protein concentration to be measured is sufficient on the order of the perfect mole. In addition, since hundreds to tens of thousands of substances can be aligned and immobilized on a single carrier, it is suitable when the types and amounts of proteins and partial peptides to be measured are large. In the same manner as in 1 above, a biological sample is added to an immobilized antibody or the like, the protein or partial peptide to be measured is bound on the microarray, and then a fluorescent or chemiluminescent substance or enzyme is bound. Add secondary antibody and incubate. Detection may be performed by adding each substrate and then measuring the fluorescent or chemiluminescent substance or visible light from the enzyme reaction.

  The present invention is digested and decomposed by a method using a microarray into a protein that exists as an intact protein in a living body in a normal state but is generated as at least one partial peptide in a living body in a state other than normal, for example, a digestion-degrading peptide. For proteins, it also includes a device for evaluating and discriminating normal and non-normal states of a living body, including detecting the type and abundance or abundance ratio of the intact protein and its partial peptide in a biological sample. . The configuration of the device is the same as the configuration of the device described in 1 above.

3. Mass Spectrometry Partial peptides such as proteins and degradation product peptides can also be analyzed by mass spectrometry using a mass spectrometer. The mass spectrometer includes a sample introduction unit, an ionization chamber, an analysis unit, a detection unit, a recording unit, and the like. Electron impact ionization (EI), chemical ionization (CI), field desorption (FD), secondary ionization (SIMS), fast atom bombardment (FAB), matrix-assisted laser desorption ionization A (MALDI) method, an electrospray ionization (ESI) method, or the like may be used. As the analysis unit, a double convergence mass spectrometer, a quadrupole mass spectrometer, a time-of-flight mass spectrometer, a Fourier transform mass spectrometer, an ion cyclotron mass spectrometer, or the like is used.

  For example, an antibody against a specific protein and its partial peptide is bound to microbeads or substrates (protein chips) that have been specially modified in advance. The microbead may be a magnetic bead. Any material can be used for the substrate. The antibodies to be used are (1) an antibody that recognizes only the full length of a specific protein, (2) an antibody that recognizes only a partial peptide, (3) an antibody that recognizes both a specific protein and its partial peptide, or A combination of (1) and (2), (1) and (3), or (2) and (3) may be used. A sample is serially diluted with a stock solution or a buffer solution, and an appropriate amount thereof is added to a microbead or a substrate to which an antibody is bound, and incubated. Thereafter, washing is performed to remove uncaptured proteins and partial peptides. The protein and partial peptide captured on the microbead or substrate are then analyzed by mass spectrometry using MALDI-TOF-MS, SELDI-TOF-MS, etc., and the peak mass number and peak intensity of each protein and partial peptide are analyzed. Measure. SELDI uses chemical functional groups on the chip surface or immobilized molecules to capture molecules with specific properties from the sample on the chip, and then removes the captured molecules by laser irradiation. This refers to a method of performing ionization / ionization. Prior to mass spectrometry, all may be cleaved into peptides by a specific proteolytic enzyme such as trypsin. The mass analysis peak (mass spectrum) and peak pattern (mass spectrum pattern) of the protein and partial peptide obtained from these measurements are analyzed by software, and the sample is evaluated and determined. The presence pattern of the intact protein and its partial peptide in the biological sample can be determined from the mass spectrum and mass spectrum pattern obtained by mass spectrometry, and a protein / partial peptide profile can be obtained.

  The present invention is digested and decomposed into a protein that exists as an intact protein in a living body in a normal state but is produced as at least one partial peptide in a living body in a state other than normal, for example, a digestion-degrading peptide, by a method using mass spectrometry. Including a device for evaluating and discriminating normal and non-normal states of a living body, including detecting the type and abundance or abundance ratio of the intact protein and its partial peptide in a biological sample. To do. The apparatus includes at least an antibody or aptamer fixing part (capturing part) and a mass spectrometer (measuring part). Furthermore, the apparatus of the present invention may include an analysis unit that analyzes the obtained data, and the analysis unit includes a data processing device and analysis software.

  In addition to the above methods, proteins and partial peptides can be analyzed by a method using two-dimensional electrophoresis, a method using surface plasmon resonance, or the like.

  In the conventional technology, only intact proteins in normal and non-normal states of the body are measured, and partial proteins generated during the synthesis process and peptides of digestion degradation products generated during the degradation process cannot be measured. It was. There was no device that could measure both intact proteins, partial proteins produced during the synthesis process, and peptides of digestion degradation products produced during the degradation process.

  The method of the present invention makes it possible to use a disease state-specific biomarker that could not be achieved conventionally for diagnosis. The means and method provided by the present invention include intact proteins in normal and non-normal states of the living body and / or partial proteins produced in the synthesis process thereof, and intact proteins and / or peptides of digestion degradation products produced in the degradation process thereof. Enables measurement.

  By using each antibody produced against the intact protein of the measurement object, the partial protein produced in the synthesis process, and the peptide of the digestion degradation product generated in the degradation process, the measurement object is selectively captured. By measuring the amount of each captured using conventional ELISA, RIA, CLIA, etc., intact proteins in normal and non-normal states of the body and partial peptides produced during the synthesis process It is now possible to measure the peptide amount or abundance ratio of digestion degradation products generated in the degradation process.

  In the system using mass spectrometry, each antibody produced against the intact protein of the measurement object, the partial protein produced during the synthesis process, and the digestion degradation product peptide produced during the degradation process is combined with a microbead or protein chip. By combining with, measurement with high sensitivity and high throughput became possible. The sensitivity of mass spectrometry using MALDI-TOF-MS or the like is usually from atto mole to zepto mole level, which is superior to ELISA, RIA, CLIA, and so on.

  The present invention provides two-dimensional electrophoresis of specimen samples collected from a subject in normal condition (a healthy person) and a subject in a condition other than normal, and compares the electrophoresis patterns of both specimen samples to obtain a normal condition. Identifies a protein, partial protein or partial peptide that is detected in, but disappears or decreases in a state other than normal, or a protein, partial protein, or partial peptide that is not detected in a normal state but detected in a state other than normal. And a method for screening a biomarker for discriminating a state other than normal. Here, the two-dimensional electrophoresis is preferably two-dimensional electrophoresis in which isoelectric focusing and SDS-PAGE are combined. Also included is a method of subjecting a specimen sample collected from a subject to two-dimensional electrophoresis and determining whether the subject is in a normal state or in a state other than normal using the presence or absence of the biomarker as an index. .

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Two-dimensional electrophoresis analysis of serum protein derived from liver cancer patient and healthy subject (1) Method 100 μg of serum protein obtained from liver cancer patient and healthy subject was analyzed by the following two-dimensional electrophoresis, respectively. .
In the first dimension isoelectric focusing, serum proteins were dissolved in a buffer containing 8M urea and run on a 13 cm immobilized pH gradient (pH 4-7) strip for 80,000 volt hours. After reductive alkylation of the protein after electrophoresis, second-dimensional SDS electrophoresis (gel concentration: 15%, gel length: 16 cm) was performed, and the protein in the gel was silver stained (Silver Staining).

(2) Results In this method, approximately 500 protein-derived spots were detected. FIG. 4 shows a pattern of serum protein derived from a liver cancer patient (HCC), and FIG. 5 shows a pattern derived from a healthy person. Those circled in the figure are serum proteins that were not detected in liver cancer patients. Of these, A is α-1 antitrypsin and B is haptoglobin. This result shows that α-1 antitrypsin and haptoglobin disappear in the serum of liver cancer patients.

Example 2 Measurement of α-1 Trypsin Inhibitor Level in Serum of Liver Cancer Patients and Healthy Persons by Enzyme Immunoassay (ELISA) (1) Method ELISA was performed using a commercially available kit and a self-made antibody. A diluted solution of anti-α-1 trypsin inhibitor antibody (primary antibody) was added to a 96-well (well) culture plate and allowed to stand at room temperature for 1 hour to adsorb the antibody to the plate. After removing the solution, a 3% serum albumin solution was added to prevent non-specific binding of the antigen to the plate and left at room temperature for 1 hour. After removal of the solution, standard serum, cancer patient serum, and healthy human serum that were appropriately serially diluted were placed in separate wells and allowed to stand at room temperature for 1 hour. Each well was washed 4 times with a buffer containing 0.1% surfactant. Further, a secondary antibody diluted solution labeled with an enzyme (peroxidase) was added thereto, and after standing for 1 hour, each well was washed 4 times. Finally, an enzyme substrate solution that develops color upon receiving the enzyme action was added and left for 30 minutes. The absorbance of each well was measured using a photometer. The amount of α-1 trypsin inhibitor in the serum of each cancer patient and healthy human serum was expressed in units by calculating the measurement results by comparing the amount of α-1 trypsin inhibitor in the standard serum with 100 units. .

  FIG. 6 is a graph showing the results. The results of experiments conducted in three ways, that is, combinations of primary antibody and secondary antibody A, B, and C, are summarized.

  A is a primary antibody and a secondary antibody of a commercially available ELISA kit, B is an antibody specific for the N-terminus of α-1 trypsin inhibitor as the primary antibody, and an antibody specific for the C-terminus is used as the secondary antibody. . In C, an antibody specific to a peptide fragment of α-1 trypsin inhibitor that is specifically observed in liver cancer patients was used as the primary and secondary antibodies. The above peptide fragment has been found by the present inventor as a biomarker candidate for liver cancer by an original method. As standard sera, commercially available pooled normal human serum was used for A and B, and pooled liver cancer patient-derived serum obtained by the present inventors was used for C.

(2) Results In this experiment, two phenomena found by the present inventor, namely (i) observed in normal persons in liver cancer patients by two-dimensional electrophoresis (live, undegraded) Almost no α-1 antitrypsin is detected (disappears). (ii) On the other hand, in the 2D-HPLC-MALDI-TOF / MS method unique to the present inventor, a specific α-1 antitrypsin peptide (SEQ ID NO: 24) is specifically detected in a liver cancer patient.

  In order to reproduce these phenomena by ELISA, the above-described methods B and C were devised by preparing primary and secondary antibodies.

  As can be seen in FIG. 6, the method using the commercially available kit of A does not show a difference between liver cancer patients and normal persons (112.6 units vs 105.7 units), but the method of B uses two methods. As a result of reflecting the result of the two-dimensional electrophoresis, that is, the amount of α-1 antitrypsin in the serum of the liver cancer patient is remarkably decreased as compared with the normal person (25.7 units vs 101.5 units). On the other hand, in the method of C, the amount of α-1 antitrypsin-derived peptide was significantly increased in liver cancer patients compared with normal individuals (99.1 units vs 24.4 units). As described above, the present inventor reduced the amount of raw α-1 antitrypsin protein that had not undergone degradation, which was found in another experiment by the inventor in liver cancer patient serum, by creating an original antibody. And succeeded in establishing a unique ELISA system that can reflect the phenomenon that specific peptides derived from the protein are increasing.

Example 3 Detection of disease biomarker peptide by immunoprecipitation / MS analysis (1) Method Immunoprecipitation
1-a) Preparation of antibody (IgG) binding gel Gel containing a polyclonal anti-α1-antitrypsin antibody (IgG) (Bethyl) and a hydrazyl group as a reactive group (Affi-Gel Hz, Bio-Rad) Were covalently bound to prepare an antibody-binding gel.

Add 150 μl of 95 mM NaIO ₄ to 500 μl (0.5 mg / ml, 0.25 mg IgG) of anti-α1-antitrypsin IgG dissolved in 20 mM sodium acetate buffer (pH 5.0) containing 0.15 M NaCl, and react for 1 hour at room temperature in the dark To cleave the cyclic structure of the sugar chain. After desalting NaIO ₄ and exchanging the buffer of IgG solution with Bio-Gel P6 DG (Bio-Rad) swollen with 0.1 M sodium acetate buffer (pH 4.5) containing 1.0 M NaCl, add 100 μl of the above gel. Then, a coupling reaction was performed at room temperature for 24 hours. Centrifugation was performed at 5,800 × g for 1 minute, and the gel bound with the antibody was collected. The amount of antibody bound was determined by measuring the absorbance of the supernatant at 280 nm and converting the concentration by the IgG extinction coefficient E ^1% _{280 nm} = 13.8 to evaluate the amount of bound antibody.

1-b) Immunoprecipitation 495 μl of 0.1% trifluoroacetic acid (TFA) was added to 5 μl of serum derived from healthy subjects, hepatitis, cirrhosis, and liver cancer patients and boiled for 10 minutes. Thereafter, the filtrate was collected by centrifugal filtration with YM10 (Millipore). 25 μl of antibody non-binding gel was added to the filtrate and incubated at 4 ° C. for 2 hours, and a supernatant from which nonspecifically adsorbed proteins and peptides were removed by centrifugation was obtained. 10 μl of antibody-binding gel was added to the supernatant, and antigen-antibody reaction was performed at 4 ° C. for 2 hours. The antibody-bound gel was precipitated by centrifugation to remove the supernatant, and the antibody-bound gel was washed with 20 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl, and then further washed with ultrapure water.

1-c) Recovery of specific binding peptide 25 μl of 0.5 M formic acid was added to the antibody binding gel and allowed to stand at room temperature for 5 minutes to release the specifically bound peptide from the antibody binding gel, and the supernatant was recovered by centrifugation.

Mass analysis with MALDI-TOF mass spectrometer
MALDI-TOF mass spectrometry is performed by adding 1 μl of specific binding peptide and 1 μl of matrix solution (10 mg / ml α-cyano-4-hydroxycinnamic acid (CHCA), 50% acetonitrile, 0.1% TFA) to the MALDI sample plate. The mass of all peptides was measured by a machine and analyzed.

(2) Results
The amount of anti-α1-antitrypsin antibody bound to Affi-Gel Hz was 1 μg IgG / μl gel.

  FIG. 7 shows the results of mass spectrometry. The mass patterns of peptides obtained from healthy subjects, hepatitis, cirrhosis, and liver cancer patients are shown in order from the top of the graph. The vertical axis represents relative intensity, and the horizontal axis represents mass.

Apart from the peptides common to all samples, disease-specific peptides were found.
Peptides common to all samples --- [2], [5], [7]
Hepatitis-specific peptide-- [3], [9]
Cirrhosis-specific peptide-- [12]
Peptide specific to liver cancer ――― [4], [11]

  Peptides derived from serum of healthy individuals, hepatitis, cirrhosis, and liver cancer patients were immunoprecipitated with anti-α1-antitrypsin antibody. In addition to peptides common to all sera ([2], [5], [7] above) Thus, peptide-derived mass spectra ([3], [9], [12], [4], [11]) specific to each patient were obtained. This result shows that different proteolytic enzymes or sets thereof are activated in each disease, and as a result, peptides specific to each disease are generated from the same protein, and these are obtained by this method. It means that it was detected. That is, this experiment revealed that different peptides derived from the same protein become disease-specific biomarkers and that they can be detected by this method.

Example 4 Search for Diagnostic Marker Peptide for Liver Disease by Two-Dimensional Liquid Chromatography (2D-LC) MALDI-TOF / MS (1) Method 475 μl of 0.1 for each 25 μl of serum from healthy individuals and patients with hepatitis, cirrhosis and liver cancer % Trifluoroacetic acid (TFA) was added and boiled at 100 ° C. for 15 minutes. Thereafter, ultrafiltration was performed using Millipore YM-10 to recover peptides with a molecular weight of 10,000 or less. The collected sample was fractionated to 1,140 per sample using two-dimensional HPLC (SCX cation exchange and C18 reverse phase column). All fractionated samples were spotted on a mass spectrometer (AXIMA CFR plus) sample platform using an online connected spotting robot (named AccuSpot), and the matrix solution (α-cyano-hydroxycinnamic acid, CHCA) was mixed and co-crystallized, and then laser irradiation was performed and mass was automatically measured in reflectron mode. Detection of differential expression peptides between healthy controls and disease samples was performed using our software, Differential Display Viewer. Peptides in which differences in expression levels were recognized were directly determined for their amino acid sequences by MS / MS and MS n-th power analysis using a mass spectrometer AXIMA QIT to identify the derived protein.

(2) Results Using the above 2D-LC MALDI-TOF / MS method, as a result of comprehensive differential analysis of peptides from serum of patients with hepatitis, cirrhosis, and liver cancer as a result of comprehensive analysis, Peptide fragments that were not detected were identified as follows.

  The lower amino acid sequence shows a set of two sequences, but the entire sequence of the first sequence shows the sequence of the constituent amino acids of the intact peptide or protein. It is a peptide fragment in which a peptide consisting of the underlined portion of the first sequence and the second sequence of each is detected. 1 represents the N-terminal.

[1] Bradykinin Intact protein in which the bradykinin-derived peptide of SEQ ID NO: 2 was specifically detected in patients with hepatitis 1
Bradykinin-derived peptide RPPGFSPF (SEQ ID NO: 2)

[2] Complement C3f Intact protein in which complement C3f-derived peptide of SEQ ID NO: 4 was specifically detected in hepatitis patients 1 SSKITHRI HW ESASLL R (SEQ ID NO: 3)
Complement C3f-derived peptide HW ESASLL (SEQ ID NO: 4)

[3] Apolipoprotein C1 Intact protein in which the apolipoprotein C1-derived peptide of SEQ ID NO: 6 could not be detected in cirrhosis patients 1 TP DVSSALDK LKEFGNTLED KARELISRIK QSELSAKM (SEQ ID NO: 5)
Apolipoprotein C1-derived peptide DVSSALDK LKEFGNTLED (SEQ ID NO: 6)

[4] Fibrinogen α chain Intact protein in which the fibrinogen α chain-derived peptide of SEQ ID NO: 8 was specifically detected in liver cancer patients
001 MFSMRIVCLV LSVVGTAWTA DSGEGDFLAE GGGVRGPRVV ERHQSACKDS
051 DWPFCSDEDW NYKCPSGCRM KGLIDEVNQD FTNRINKLKN SLFEYQKNNK
101 DSHSLTTNIM EILRGDFSSA NNRDNTYNRV SEDLRSRIEV LKRKVIEKVQ
151 HIQLLQKNVR AQLVDMKRLE VDIDIKIRSC RGSCSRALAR EVDLKDYEDQ
201 QKQLEQVIAK DLLPSRDRQH LPLIKMKPVP DLVPGNFKSQ LQKVPPEWKA
251 LTDMPQMRME LERPGGNEIT RGGSTSYGTG SETESPRNPS SAGSWNSGSS
301 GPGSTGNRNP GSSGTGGTAT WKPGSSGPGS TGSWNSGSSG TGSTGNQNPG
351 SPRPGSTGTW NPGSSERGSA GHWTSESSVS GSTGQWHSES GSFRPDSPGS
401 GNARPNNPDW GTFEEVSGNV SPGTRREYHT EKLVTSKGDK ELRTGKEKVT
451 SGSTTTTRRS CSKTVTKTVI GPDGHKEVTK EVVTSEDGSD CPEAMDLGTL
501 SGIGTLDGFR HRHPDEAAFF DTASTGKTFP GFFSPMLGEF VSETESR GSE
551 SGIFTNTKES SSHHPGIAEF PSRG KSSSYS KQFTSSTSYN RGDSTFESKS
601 YKMADEAGSE ADHEGTHSTK RGHAKSRPVR GIHTSPLGKP SLSP (SEQ ID NO: 7)

Fibrinogen alpha chain-derived peptide
GSESGIFTNTKESSSHHPGIAEFPSRG (SEQ ID NO: 8)

[5] Fibrinopeptide A like An intact protein in which the fibrinopeptide A like-derived peptide of SEQ ID NO: 9 could not be detected in liver cancer patients
001 MFSMRIVCLV LSVVGTAWTA DSGEGDFLAE GGGV RGPRVV ERHQSACKDS
051 DWPFCSDEDW NYKCPSGCRM KGLIDEVNQD FTNRINKLKN SLFEYQKNNK
101 DSHSLTTNIM EILRGDFSSA NNRDNTYNRV SEDLRSRIEV LKRKVIEKVQ
151 HIQLLQKNVR AQLVDMKRLE VDIDIKIRSC RGSCSRALAR EVDLKDYEDQ
201 QKQLEQVIAK DLLPSRDRQH LPLIKMKPVP DLVPGNFKSQ LQKVPPEWKA
251 LTDMPQMRME LERPGGNEIT RGGSTSYGTG SETESPRNPS SAGSWNSGSS
301 GPGSTGNRNP GSSGTGGTAT WKPGSSGPGS TGSWNSGSSG TGSTGNQNPG
351 SPRPGSTGTW NPGSSERGSA GHWTSESSVS GSTGQWHSES GSFRPDSPGS
401 GNARPNNPDW GTFEEVSGNV SPGTRREYHT EKLVTSKGDK ELRTGKEKVT
451 SGSTTTTRRS CSKTVTKTVI GPDGHKEVTK EVVTSEDGSD CPEAMDLGTL
501 SGIGTLDGFR HRHPDEAAFF DTASTGKTFP GFFSPMLGEF VSETESRGSE
551 SGIFTNTKES SSHHPGIAEF PSRGKSSSYS KQFTSSTSYN RGDSTFESKS
601 YKMADEAGSE ADHEGTHSTK RGHAKSRPVR GIHTSPLGKP SLSP (SEQ ID NO: 9)

Fibrinopeptide A like peptide
DSGEGDFLAEGGGV (SEQ ID NO: 10)

[6] Complement C4A An intact protein in which the complement C4A-derived peptide of SEQ ID NO: 12 was detected in patients with liver cancer and cirrhosis
0001 MRLLWGLIWA SSFFTLSLQK PRLLLFSPSV VHLGVPLSVG VQLQDVPRGQ
0051 VVKGSVFLRN PSRNNVPCSP KVDFTLSSER DFALLSLQVP LKDAKSCGLH
0101 QLLRGPEVQL VAHSPWLKDS LSRTTNIQGI NLLFSSRRGH LFLQTDQPIY
0151 NPGQRVRYRV FALDQKMRPS TDTITVMVEN SHGLRVRKKE VYMPSSIFQD
0201 DFVIPDISEP GTWKISARFS DGLESNSSTQ FEVKKYVLPN FEVKITPGKP
0251 YILTVPGHLD EMQLDIQARY IYGKPVQGVA YVRFGLLDED GKKTFFRGLE
0301 SQTKLVNGQS HISLSKAEFQ DALEKLNMGI TDLQGLRLYV AAAIIEYPGG
0351 EMEEAELTSW YFVSSPFSLD LSKTKRHLVP GAPFLLQALV REMSGSPASG
0401 IPVKVSATVS SPGSVPEVQD IQQNTDGSGQ VSIPIIIPQT ISELQLSVSA
0451 GSPHPAIARL TVAAPPSGGP GFLSIERPDS RPPRVGDTLN LNLRAVGSGA
0501 TFSHYYYMIL SRGQIVFMNR EPKRTLTSVS VFVDHHLAPS FYFVAFYYHG
0551 DHPVANSLRV DVQAGACEGK LELSVDGAKQ YRNGESVKLH LETDSLALVA
0601 LGALDTALYA AGSKSHKPLN MGKVFEAMNS YDLGCGPGGG DSALQVFQAA
0651 GLAFSDGDQW TLSRKRLSCP KEKTTRKKRN VNFQKAINEK LGQYASPTAK
0701 RCCQDGVTRL PMMRSCEQRA ARVQQLDCRE PFLSCCQFAE SLRKKSRDKG
0751 QAGLQRALEI LQEEDLIDED DIPVRSFFPE NWLWRVETVD RFQILTLWLP
0801 DSLTTWEIHG LSLSKTKGLC VATPVQLRVF REFHLHLRLP MSVRRFEQLE
0851 LRPVLYNYLD KNLTVSVHVS PVEGLCLAGG GGLAQQVLVP AGSARPVAFS
0901 VVPTAAAAVS LKVVARGSFE FPVGDAVSKV LQIEKEGAIH REELVYELNP
0951 LDHRGR TLEI PGNSDPNMIP DGDFNSYVRV TASD PLDTLG SEGALSPGGV
1001 ASLLRLPRGC GEQTMIYLAP TLAASRYLDK TEQWSTLPPE TKDHAVDLIQ
1051 KGYMRIQQFR KADGSYAAWL SRDSSTWLTA FVLKVLSLAQ EQVGGSPEKL
1101 QETSNWLLSQ QQADGSFQDP CPVLDRSMQG GLVGNDETVA LTAFVTIALH
1151 HGLAVFQDEG AEPLKQRVEA SISKANSFLG EKASAGLLGA HAAAITAYAL
1201 TLTKAPVDLL GVAHNNLMAM AQETGDNLYW GSVTGSQSNA VSPTPAPRNP
1251 SDPMPQAPAL WIETTAYALL HLLLHEGKAE MADQAAAWLT RQGSFQGGFR
1301 STQDTVIALD ALSAYWIASH TTEERGLNVT LSSTGRNGFK SHALQLNNRQ
1351 IRGLEEELQF SLGSKINVKV GGNSKGTLKV LRTYNVLDMK NTTCQDLQIE
1401 VTVKGHVEYT MEANEDYEDY EYDELPAKDD PDAPLQPVTP LQLFEGRRNR
1451 RRREAPKVVE EQESRVHYTV CIWRNGKVGL SGMAIADVTL LSGFHALRAD
1501 LEKLTSLSDR YVSHFETEGP HVLLYFDSVP TSRECVGFEA VQEVPVGLVQ
1551 PASATLYDYY NPERRCSVFY GAPSKSRLLA TLCSAEVCQC AEGKCPRQRR
1601 ALERGLQDED GYRMKFACYY PRVEYGFQVK VLREDSRAAF RLFETKITQV
1651 LHFTKDVKAA ANQMRNFLVR ASCRLRLEPG KEYLIMGLDG ATYDLEGHPQ
1701 YLLDSNSWIE EMPSERLCRS TRQRAACAQL NDFLQEYGTQ GCQV
(SEQ ID NO: 11)

Complement C4A-derived peptide
TLEIPGNSDPNMIPDGDFNSYVRVTASD (SEQ ID NO: 12)

[7] Inter-α trypsin inhibitor Intact protein in which peptide derived from inter-α trypsin inhibitor of SEQ ID NO: 14 was detected in patients with liver cancer and hepatitis
001 MKPPRPVRTC SKVLVLLSLL AIHQTTTAEK NGIDIYSLTV DSRVSSRFAH
051 TVVTSRVVNR ANTVQEATFQ MELPKKAFIT NFSMNIDGMT YPGIIKEKAE
101 AQAQYSAAVA KGKSAGLVKA TGRNMEQFQV SVSVAPNAKI TFELVYEELL
151 KRRLGVYELL LKVRPQQLVK HLQMDIHIFE PQGISFLETE STFMTNQLVD
201 ALTTWQNKTK AHIRFKPTLS QQQKSPEQQE TVLDGNLIIR YDVDRAISGG
251 SIQIENGYFV HYFAPEGLTT MPKNVVFVID KSGSMSGRKI QQTREALIKI
301 LDDLSPRDQF NLIVFSTEAT QWRPSLVPAS AENVNKARSF AAGIQALGGT
351 NINDAMLMAV QLLDSSNQEE RLPEGSVSLI ILLTDGDPTV GETNPRSIQN
401 NVREAVSGRY SLFCLGFGFD VSYAFLEKLA LDNGGLARRI HEDSDSALQL
451 QDFYQEVANP LLTAVTFEYP SNAVEEVTQN NFRLLFKGSE MVVAGKLQDR
501 GPDVLTATVS GKLPTQNITF QTESSVAEQE AEFQSPKYIF HNFMERLWAY
551 LTIQQLLEQT VSASDADQQA LRNQALNLSL AYSFVTPLTS MVVTKPDDQE
601 QSQVAEKPME GESRNRNVHS GSTFFKYYLQ GAKIPKPEAS FSPRRGWNRQ
651 AGAAGSRMNF RPGVLSSRQL GLPGPPD VPD HAAYHPF RRL AILPASAPPA
701 TSNPDPAVSR VMNMKIEETT MTTQTPAPIQ APSAILPLPG QSVERLCVDP
751 RHRQGPVNLL SDPEQGVEVT GQYEREKAGF SWIEVTFKNP LVWVHASPEH
801 VVVTRNRRSS AYKWKETLFS VMPGLKMTMD KTGLLLLSDP DKVTIGLLFW
851 DGRGEGLRLL LRDTDRFSSH VGGTLGQFYQ EVLWGSPAAS DDGRRTLRVQ
901 GNDHSATRER RLDYQEGPPG VEISCWSVEL (SEQ ID NO: 13)

Inter alpha trypsin inhibitor-derived peptide
VPDHAAYHPF (SEQ ID NO: 14)

[8] Gelsolin An intact protein in which the gelsolin-derived peptide of SEQ ID NO: 16 could not be detected in liver cancer patients
001 MAPHRPAPAL LCALSLALCA LSLPVRAATA SRGASQAGAP QGRVPEARPN
051 SMVVEHPEFL KAGKEPGLQI WRVEKFDLVP VPTNLYGDFF TGDAYVILKT
101 VQLRNGNLQY DLHYWLGNEC SQDESGAAAI FTVQLDDYLN GRAVQHREVQ
151 GFESATFLGY FKSGLKYKKG GVASGFKHVV PNEVVVQRLF QVKGRRVVRA
201 TEVPVSWESF NNGDCFILDL GNNIHQWCGS NSNRYERLKA TQVSKGIRDN
251 ERSGRARVHV SEEGTEPEAM LQVLGPKPAL PAGTEDTAKE DAANRKLAKL
301 YKVSNGAGTM SVSLVADENP FAQGALKSED CFILDHGKDG KIFVWKGKQA
351 NTEERKAALK TASDFITKMD YPKQTQVSVL PEGGETPLFK QFFKNWRDPD
401 QTD GLGLSYL SSHIANVERV PFD AATLHTS TAMAAQHGMD DDGTGQKQIW
451 RIEGSNKVPV DPATYGQFYG GDSYIILYNY RHGGRQGQII YNWQGAQSTQ
501 DEVAASAILT AQLDEELGGT PVQSRVVQGK EPAHLMSLFG GKPMIIYKGG
551 TSREGGQTAP ASTRLFQVRA NSAGATRAVE VLPKAGALNS NDAFVLKTPS
601 AAYLWVGTGA SEAEKTGAQE LLRVLRAQPV QVAEGSEPDG FWEALGGKAA
651 YRTSPRLKDK KMDAHPPRLF ACSNKIGRFV IEEVPGELMQ EDLATDDVML
701 LDTWDQVFVW VGKDSQEEEK TEALTSAKRY IETDPANRDR RTPITVVKQG
751 FEPPSFVGWF LGWDDDYWSV DPLDRAMAEL AA (SEQ ID NO: 15)

Gelsolin derived peptide
GLGLSYLSSHIANVERVPFD (SEQ ID NO: 16)

[9] Apolipoprotein A1 Intact protein in which the apolipoprotein A1-derived peptide of SEQ ID NO: 18 could not be detected in liver cancer patients
1 DEPPQSPWDR VKDLATVYVD VLKDSG (SEQ ID NO: 17)
Apolipoprotein A1-derived peptide
DEPPQSPWDRVKDLATVYVD (SEQ ID NO: 18)

[10] α2 Macroglobulin An intact protein in which the α2 macroglobulin-derived peptide of SEQ ID NO: 20 was detected in patients with liver cancer, cirrhosis and hepatitis
0001 MGLLMSRVQY RLESIWSPPA PSFCNMGKNK LLHPSLVLLL LVLLPTDASV
0051 SGKPQYMVLV PSLLHTETTE KGCVLLSYLN ETVTVSASLE SVRGNRSLFT
0101 DLEAENVVLH CVAFAVPKSS SNEEVMFLTV QVKGPTQEFK KRTTVMVKNE
0151 DSLVFVQTDK SIYKPGQTVK FRVVSMDENF HPLNELIPLV YIQDPKGNRI
0201 AQWQSFQLEG GLKQFSFPLS SEPFQGSYKV VVQKKSGGRT EHPFTVEEFV
0251 LPKFEVQVTV PKIITILEEE MNVSVCGLYT YGKPVPGHVT VSICRKYSDA
0301 SDCHGEDSQA FCEKFSGQLN SHGCFYQQVK TKVFQLKRKE YEMKLHTEAQ
0351 IQEEGTVVEL TGRQSSEITR TITKLSFVKV DSHFRQGIPF FGQVRLVDGK
0401 GVPIPNKVIF IRGNEANYYS NATTDEHGLV QFSINTTNVM GTSLTVRVNY
0451 KDRSPCYGYQ WVSEEHEEAH HTAYLVFSPS KSFVHLEPMS HELPCGHTQT
0501 VQAHYILNGG TLLGLKKLSF YYLIMAKGGI VRTGTHGLLV KQEDMKGHFS
0551 ISIPVKSDIA PVARLLIYAV LPTGDVIGDS AKYDVENCLA NKVDLSFSPS
0601 QSLPASHAHL RVTAAPQSVC ALRAVDQSVL LMKPDAELSA SSVYNLLPEK
0651 DLTGFPGPLN DQDDEDCINR HNVYINGITY TPVSSTNEKD MYSFLEDMGL
0701 KAFTNSKIRK PKMCPQLQQY EMHGPEGLRV GFYESDVMGR GHARLVHVEE
0751 PHTETVRKYF PETWIWDLVV VNSAGVAEVG VTVPDTITEW KAGAFCLSED
0801 AGLGISSTAS LRAFQPFFVE LTMPYSVIRG EAFTLKATVL NYLPKCIRVS
0851 VQLEASPAFL AVPVEKEQAP HCICANGRQT VSWAVTPKSL GNVNFTVSAE
0901 ALESQELCGT EVPSVPEHGR KDTVIKPLLV EPEGLEKETT FNSLLCPSGG
0951 EVSEELSLKL PPNVVEESAR ASVSVLGDIL GSAMQNTQNL LQMPYGCGEQ
1001 NMVLFAPNIY VLDYLNETQQ LTPEVKSKAI GYLNTGYQRQ LNYKHYDGSY
1051 STFGERYGRN QGNTWLTAFV LKTFAQARAY IFIDEAHITQ ALIWLSQRQK
1101 DNGCFRSSGS LLNNAIKGGV EDEVTLSAYI TIALLEIPLT VTHPVVRNAL
1151 FCLESAWKTA QEGDHGSHVY TKALLAYAFA LAGNQDKRKE VLKSLNEEAV
1201 KKDNSVHWER PQKPKAPVGH FYEPQAPSAE VEMTSYVLLA YLTAQPAPTS
1251 EDLTSATNIV KWITKQQNAQ GGFSSTQDTV VALHALSKYG AATFTRTGKA
1301 AQVTIQSSGT FSSKFQVDNN NRLLLQQVSL PELPGEYSMK VTGEGCVYLQ
1351 TSLKYNILPE KEEFPFALGV QTLPQTCDEP KAHTSFQISL SVSYTGSRSA
1401 SNMAIVDVKM VSGFIPLKPT VKMLERSNHV SRTEVSSNHV LIYLDKVSNQ
1451 TLSLFFTVLQ D VPVRDLKPA IVKVYD YYET DEFAIAEYNA PCSKDLGNA
(SEQ ID NO: 19)

α2 Macroglobulin derived peptide
VPVRDLKPAIVKVYD (SEQ ID NO: 20)

[11] Fibrinogen α chain Intact protein in which the fibrinogen α chain-derived peptide of SEQ ID NO: 22 was detected in liver cancer patients
001 MFSMRIVCLV LSVVGTAWTA DSGEGDFLAE GGGVRGPRVV ERHQSACKDS
051 DWPFCSDEDW NYKCPSGCRM KGLIDEVNQD FTNRINKLKN SLFEYQKNNK
101 DSHSLTTNIM EILRGDFSSA NNRDNTYNRV SEDLRSRIEV LKRKVIEKVQ
151 HIQLLQKNVR AQLVDMKRLE VDIDIKIRSC RGSCSRALAR EVDLKDYEDQ
201 QKQLEQVIAK DLLPSRDRQH LPLIKMKPVP DLVPGNFKSQ LQKVPPEWKA
251 LTDMPQMRME LERPGGNEIT RGGSTSYGTG SETESPRNPS SAGSWNSGSS
301 GPGSTGNRNP GSSGTGGTAT WKPGSSGPGS TGSWNSGSSG TGSTGNQNPG
351 SPRPGSTGTW NPGSSERGSA GHWTSESSVS GSTGQWHSES GSFRPDSPGS
401 GNARPNNPDW GTFEEVSGNV SPGTRREYHT EKLVTSKGDK ELRTGKEKVT
451 SGSTTTTRRS CSKTVTKTVI GPDGHKEVTK EVVTSEDGSD CPEAMDLGTL
501 SGIGTLDGFR HRHPDEAAFF DTASTGKTFP GFFSPMLGEF VSETESR GSE
551 SGIFTNTKES SSHHPGIAEF PSRG KSSSYS KQFTSSTSYN RGDSTFESKS
601 YKMADEAGSE ADHEGTHSTK RGHAKSRPVR GIHTSPLGKP SLSP
(SEQ ID NO: 21)

Fibrinogen alpha chain-derived peptide
GSESGIFTNTKESSSHHPGIAEFPSRG (SEQ ID NO: 22)

[12] α1 antitrypsin An intact protein in which the α1 antitrypsin-derived peptide of SEQ ID NO: 24 was specifically detected in patients with liver cancer, cirrhosis or hepatitis
001 FNKITPNLAE FAFSLYRQLA HQSNSTNIFF SPVSIATAFA MLSLGTKADT
051 HDEILEGLNF NLTEIPEAQI HEGFQELLRT LNQPDSQLQL TTGNGLFLSE
101 GLKLVDKFLE D VKKLYHSEA FTVNFGD TEE AKKQINDYVE KGTQGKIVDL
151 VKELDRDTVF ALVNYIFFKG KWERPFEVKD TEEEDFHVDQ VTTVKVPMMK
201 RLGMFNIQHC KKLSSWVLLM KYLGNATAIF FLPDEGKLQH LENELTHDII
251 TKFLENEDRR SASLHLPKLS ITGTYDLKSV LGQLGITKVF SNGADLSGVT
301 EEAPLKLSKA VHKAVLTIDE KGTEAAGAMF LEAIPMSIPP EVKFNKPFVF
351 LMIEQNTKSP LFMGKVVNPT QK (SEQ ID NO: 23)

α1-antitrypsin-derived protein
VKKLYHSEAFTVNFGD (SEQ ID NO: 24)

[13] Inter-α trypsin inhibitor heavy chain H4 precursor Intact protein in which the peptide derived from inter-α trypsin inhibitor heavy chain H4 precursor of SEQ ID NO: 26 was specifically detected in patients with liver cancer and hepatitis
001 MKPPRPVRTC SKVLVLLSLL AIHQTTTAEK NGIDIYSLTV DSRVSSRFAH
051 TVVTSRVVNR ANTVQEATFQ MELPKKAFIT NFSMNIDGMT YPGIIKEKAE
101 AQAQYSAAVA KGKSAGLVKA TGRNMEQFQV SVSVAPNAKI TFELVYEELL
151 KRRLGVYELL LKVRPQQLVK HLQMDIHIFE PQGISFLETE STFMTNQLVD
201 ALTTWQNKTK AHIRFKPTLS QQQKSPEQQE TVLDGNLIIR YDVDRAISGG
251 SIQIENGYFV HYFAPEGLTT MPKNVVFVID KSGSMSGRKI QQTREALIKI
301 LDDLSPRDQF NLIVFSTEAT QWRPSLVPAS AENVNKARSF AAGIQALGGT
351 NINDAMLMAV QLLDSSNQEE RLPEGSVSLI ILLTDGDPTV GETNPRSIQN
401 NVREAVSGRY SLFCLGFGFD VSYAFLEKLA LDNGGLARRI HEDSDSALQL
451 QDFYQEVANP LLTAVTFEYP SNAVEEVTQN NFRLLFKGSE MVVAGKLQDR
501 GPDVLTATVS GKLPTQNITF QTESSVAEQE AEFQSPKYIF HNFMERLWAY
551 LTIQQLLEQT VSASDADQQA LRNQALNLSL AYSFVTPLTS MVVTKPDDQE
601 QSQVAEKPME GESRNRNVHS GSTFFKYYLQ GAKIPKPEAS FSPRRGWNRQ
651 AGAAGSRMNF RPGVLSSRQL GLPGPPDVPD HAAYHPFR RL AILPASAPPA
701 TSNPD PAVSR VMNMKIEETT MTTQTPAPIQ APSAILPLPG QSVERLCVDP
751 RHRQGPVNLL SDPEQGVEVT GQYEREKAGF SWIEVTFKNP LVWVHASPEH
801 VVVTRNRRSS AYKWKETLFS VMPGLKMTMD KTGLLLLSDP DKVTIGLLFW
851 DGRGEGLRLL LRDTDRFSSH VGGTLGQFYQ EVLWGSPAAS DDGRRTLRVQ
901 GNDHSATRER RLDYQEGPPG VEISCWSVEL (SEQ ID NO: 25)

Inter alpha trypsin inhibitor heavy chain H4 precursor-derived peptide
RLAILPASAPPATSNPD (SEQ ID NO: 26)

The partial peptide was present with a sugar chain added as shown below.
RLAILPASAPPATSNPD + -GlcNAc-Hex-GlcNAc-Hex

[14] Inter alpha trypsin inhibitor heavy chain H4 precursor intact protein
001 MKPPRPVRTC SKVLVLLSLL AIHQTTTAEK NGIDIYSLTV DSRVSSRFAH
051 TVVTSRVVNR ANTVQEATFQ MELPKKAFIT NFSMNIDGMT YPGIIKEKAE
101 AQAQYSAAVA KGKSAGLVKA TGRNMEQFQV SVSVAPNAKI TFELVYEELL
151 KRRLGVYELL LKVRPQQLVK HLQMDIHIFE PQGISFLETE STFMTNQLVD
201 ALTTWQNKTK AHIRFKPTLS QQQKSPEQQE TVLDGNLIIR YDVDRAISGG
251 SIQIENGYFV HYFAPEGLTT MPKNVVFVID KSGSMSGRKI QQTREALIKI
301 LDDLSPRDQF NLIVFSTEAT QWRPSLVPAS AENVNKARSF AAGIQALGGT
351 NINDAMLMAV QLLDSSNQEE RLPEGSVSLI ILLTDGDPTV GETNPRSIQN
401 NVREAVSGRY SLFCLGFGFD VSYAFLEKLA LDNGGLARRI HEDSDSALQL
451 QDFYQEVANP LLTAVTFEYP SNAVEEVTQN NFRLLFKGSE MVVAGKLQDR
501 GPDVLTATVS GKLPTQNITF QTESSVAEQE AEFQSPKYIF HNFMERLWAY
551 LTIQQLLEQT VSASDADQQA LRNQALNLSL AYSFVTPLTS MVVTKPDDQE
601 QSQVAEKPME GESRNRNVHS GSTFFKYYLQ GAKIPKPEAS FSPRRGWNRQ
651 AGAAGSRMNF RPGVLSSRQL GLPGPPD VPD HAAYHPF RRL AILPASAPPA
701 TSNPDPAVSR VMNMKIEETT MTTQTPAPIQ APSAILPLPG QSVERLCVDP
751 RHRQGPVNLL SDPEQGVEVT GQYEREKAGF SWIEVTFKNP LVWVHASPEH
801 VVVTRNRRSS AYKWKETLFS VMPGLKMTMD KTGLLLLSDP DKVTIGLLFW
851 DGRGEGLRLL LRDTDRFSSH VGGTLGQFYQ EVLWGSPAAS DDGRRTLRVQ
901 GNDHSATRER RLDYQEGPPG VEISCWSVEL (SEQ ID NO: 25)

Inter alpha trypsin inhibitor heavy chain H4 precursor-derived peptide
VPDHAAYHPF (SEQ ID NO: 27)

In addition, the same study was performed on patients with toxemia of pregnancy, and partial peptides of the following proteins were detected.
[15] Heparin cofactor II An intact protein in which the heparin cofactor II-derived peptide of SEQ ID NO: 29 was detected in pregnancy toxemia
001 MKHSLNALLI FLIITSAWG G SKGPLDQLEK GGETAQSAD P QWEQLNNKNL
051 SMPLLPADFH KENTVTNDWI PEGEEDDDYL DLEKIFSEDD DYIDIVDSLS
101 VSPTDSDVSA GNILQLFHGK SRIQRLNIVN AKFAFNLYRV LKDQVNTFDN
151 IFIAPVGIST AMGMISLGLK GETHEQVHSI LHFKDFVNAS SKYEITTIHN
201 LFRKLTHRLF RRNFGYTLRS VNDLYIQKQF PILLDFKTKV REYYFAEAQI
251 ADFSDPAFIS KTNNHIMKLT KGLIKDALEN IDPATQMMIL NCIYFKGSWV
301 NKFPVEMTHN HNFRLNEREV VKVSMMQTKG NFLAANDQEL DCDILQLEYV
351 GGISMLIVVP HKMSGMKTLE AQLTPGVVER WQKSMTNRTR EVLLPKFKLE
401 KNYNLVESLK LMGIRMLFDK NGNMAGISDQ RIAIDLFKHQ GTITVNEEGT
451 QATTVTTVGF MPLSTQVRFT VDRPFLFLIY EHRTSCLLFM GRVANPSRS
(SEQ ID NO: 28)

Heparin cofactor II-derived peptide
GSKGPLDQLEK GGETAQSAD (SEQ ID NO: 29)

  In this experiment, using the two-dimensional liquid chromatography (2D-LC) MALDI-TOF / MS method originally developed by the inventor, we comprehensively cover peptides derived from sera of healthy subjects and hepatitis, cirrhosis, and liver cancer patients. Through analysis, peptides that appear or disappear in a disease-specific manner could be identified. All of these were fragmented peptides that appeared to have been cleaved by their specific proteolytic enzymes. That is, this experiment showed that a fragment peptide produced by a disease-specific activated proteolytic enzyme present in patient serum can be a disease-specific biomarker. It was also revealed that the analysis system newly developed by the present inventor can detect such peptides.

Example 5 Analysis using SELDI-TOF-MS (1) Method
Using SELDI-TOF-MS (Cyphergen Biosystems), sera from patients with chronic hepatitis, cirrhosis, and hepatocellular carcinoma were measured against normal samples versus other disease samples.

As a pretreatment, Biomek200 (BECKMAN COULTER) was used, and the serum was denatured by mixing with 9 M urea at a ratio of 1: 1.5 at 4 ° C. for 20 minutes. This sample was mixed with 1 M urea buffer at a ratio of 1: 1 and applied to an anion exchange resin (Q CERAMIC HYPER DF). After stirring at 4 ° C. for 30 minutes, fractionation was performed with each buffer in the order of pH 9, 7, 5, 4, 3, and organic solvent. (pH 9: 50 mM Tris-HCl / 0.1% OGP ^* , pH 7: 50 mM HEPES / 0.1% OGP, pH 5: 100 mM sodium acetate / 0.1% OGP, pH 4: 100 mM sodium acetate / 0.1% OGP pH 3: 50 mM sodium citrate / 0.1% OGP, organic solvent: 33.3% isopropanol / 16.7% acetonitrile / 0.1% TFA) ^* OGP: 1-On-octyl-β-D-glucopyranoside. This fraction sample and binding buffer (0.1 M sodium acetate pH 4) were assayed at a ratio of 1: 9 to CM10 on the protein chip. Washing was performed 3 times using 150 μl of washing buffer (0.1 M acetate buffer pH 4), rinsing was performed twice with 200 μl of milli-Q, and 1 μl of 50% SPA was applied twice when air-dried.

  The protein chip used is CM10, and the surface of the substrate to be measured is modified with a functional group (carboxymethyl) having intestinal ion exchange properties.

(2) Results FIG. 8 shows serum protein profiling by gel viewer. FIG. 8 shows the results of analyzing sera from normal healthy subjects, chronic hepatitis C, hepatic cirrhosis, and hepatocellular carcinoma patients in duplicate using the SELDI-TOF-MS method. It is the result of analyzing normal control versus disease with CM10. Top 4 normal samples, 6 chronic hepatitis samples, 3 cirrhosis (LC) samples, 3 hepatocellular carcinoma (HCC) samples. In FIG. 8, the display is obtained by converting the intensity of the peak obtained by the mass spectrometer into a heat map. As indicated by the arrows, peptides that were not found normally were found in patients with chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Some peptides disappeared due to cirrhosis.

  Three peaks between 8000 and 9000 Da indicated by arrows are detected in chronic hepatitis, cirrhosis, and hepatocyte cancer, but not in normal. From the above, the three peaks are one of the biomarker supplements for this disease.

  Serum protein profiling by gelview is shown. It is the result of analyzing normal control versus disease with CM1O. Similarly, FIG. 9 shows the results of a single test performed with a double test. From the top, 4 normal samples, 2 chronic hepatitis (NR) samples, 2 chronic hepatitis (VR) samples, and 4 cirrhosis (LC) samples. , 4 samples of hepatocellular carcinoma (HCC). A peak between 15000 and 16000 Da indicated by a red arrow is detected in chronic hepatitis (VR) but not in chronic hepatitis (NR). From the above, this peak is judged to be a marker indicating the effect of IFN on chronic hepatitis.

Example 6 Analysis of serum protein of chronic hepatitis and cirrhosis by two-dimensional electrophoresis; Discovery of novel liver disease marker protein and discrimination of chronic hepatitis and cirrhosis by multi-marker (1) Method 19 healthy individuals and 24 chronic hepatitis patients, 100 μg of serum protein obtained from each of 20 patients with cirrhosis was analyzed by two-dimensional electrophoresis as follows.

  First-dimension isoelectric focusing is performed by dissolving serum proteins in a buffer containing 8M urea 2% detergent CHAPS and running on 80,000 volt hours on a 13 cm immobilized pH gradient (pH 4-7) strip. did. After electrophoresis, the proteins in the gel were reductively alkylated with 2% SDS buffer solution containing DTT and iodoacetamide, respectively, followed by second-dimensional SDS electrophoresis (gel concentration 15%, gel length 16 cm). The protein was silver stained (Silver Staining kit, Amersham).

(2) Results In this example, approximately 500 protein-derived spots were detected. FIG. 10 shows an example of an electrophoresis pattern of a healthy human-derived serum protein, and FIG. 10 shows an example of an electrophoresis pattern of a liver cirrhosis patient-derived serum protein. In the electrophoresis pattern diagram, the presence or absence of detection of spots A, B, C, D, and E circled varies from sample to sample. A was detected in all sera from patients with hepatitis and cirrhosis. On the other hand, spots B, C, D, and E were detected in almost all healthy subjects in the majority of hepatitis patients, but were not detected in many samples in patients with cirrhosis. These spots are cut out, the protein in the gel is treated with trypsin (Promega) overnight to make peptide fragments, desalted using C18 Tip (Eppendorf), and CHCA (α-cyano-hydroxy as matrix) (Cinnamic acid) was added and co-crystallized. This was applied to a mass spectrometer (AXIMA-CFR, Shimadzu Corporation) to obtain a mass pattern of the above peptide, and analyzed with the protein identification software Mascot. As a result, as shown in FIG. 10, A was found to be transthyretin (tetramer), B was α-1 antitrypsin, C was haptoglobin α2 chain, D was transthyretin (monomer), and E was haptoglobin β chain. . In Table 1, the result regarding the presence or absence of the detection of the spot AE of 19 healthy persons, 24 cases of chronic hepatitis, and 20 cases of cirrhosis was collectively shown (Table 1).

  In the table, 1 indicates that the spot was detected at a normal level, and 0 indicates that the spot was not detected or could be detected but had a very low intensity. Moreover, the symbol below the column of healthy persons, chronic hepatitis and cirrhosis in the table indicates the specimen sample number.

  In this experiment, the following two findings were found regarding serum protein markers for liver disease. Spot A on the two-dimensional electrophoresis gel pattern detected in this experiment is a liver disease marker protein that distinguishes chronic hepatitis patients and cirrhosis patients from healthy individuals. Patients with cirrhosis with a probability of 90% or more in both sensitivity and specificity by combining the presence or absence of detection of spots B, C, D, and E on the two-dimensional electrophoresis gel pattern detected in this experiment as a multimarker Can be distinguished from patients with chronic hepatitis. This new identification method found in this experiment was named BE test.

In the BE test, the proportion of each sample serum that could not detect each spot was as follows (n = 24, 20).
B; chronic hepatitis 4%, cirrhosis 60%
C; chronic hepatitis 17%, cirrhosis 60%
D; Chronic hepatitis 13%, cirrhosis 40%
E; chronic hepatitis 4%, cirrhosis 75%

  From this result, cirrhosis can be detected with sensitivity of 75% and specificity of 96%, using E as an index.

  Of the spots B, C, D, and E, two or more spots were not detected in one of 24 patients with chronic hepatitis and 17 of 20 patients with cirrhosis. That is, cirrhosis can be detected with a sensitivity of 90% and a specificity of 96%, using as an index that two or more spots among spots B, C, D, and E are not detected. Of these, the sensitivity is 90% and specificity is 92%, with B or E not being detected as an index, and the sensitivity is 95% and specificity is 80% with D and E being 90% and specificity being not detected. It is possible to detect cirrhosis with sensitivity of 85% and specificity of 88% using as an index that no is detected.

It is a figure which shows the concept of the method of this invention, and is a figure which shows the protein / partial peptide profile in states other than normal. It is a figure which shows the concept of the method of this invention, and is a figure which shows the mechanism in which a partial peptide produces | generates by intact digestive decomposition from intact protein. It is a figure which shows the concept of the method of this invention, and is a figure which shows the mechanism in which a partial peptide is produced | generated by digestion decomposition | disassembly in a cell from intact protein. It is a figure which shows the result of the two-dimensional electrophoresis analysis of a liver cancer patient-derived serum protein. It is a figure which shows the result of the two-dimensional electrophoresis analysis of a healthy person-derived serum protein. It is a figure which shows the result of the measurement of the amount of alpha-1 trypsin inhibitor in a liver cancer patient and healthy subject serum by enzyme immunity method (ELISA). It is a figure which shows the result of the mass spectrometry showing the disease biomarker peptide detected by the immunoprecipitation method / MS analysis. It is a figure which shows the protein / partial peptide profile of the serum of the chronic hepatitis of a liver cirrhosis, a hepatoma, and the serum of a normal person measured by mass spectrometry. It is a figure which shows the protein / partial peptide profile of the serum of the chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma patient measured by mass spectrometry, and the serum of a normal person (the 2). It is a figure which shows the two-dimensional electrophoresis pattern of a healthy person origin serum protein and a liver cirrhosis patient origin serum protein.

Claims (8)

A test method for evaluating and discriminating a normal state and a non-normal state of a living body, a disease state, or a progress degree of a disease state using at least one of a protein, a partial protein of the protein and a partial peptide of the protein as a marker, said protein is present as an intact protein in vivo in a normal state, which is present as at least one partial protein or partial peptide of the intact protein in vivo in a state other than the normal, a said intact in serum protein, the partial protein and / or the type of the partial peptide, to measure the abundance and / or abundance, saw including obtaining a protein / partial peptide profile, wherein the protein to be measured, partial protein and / or parts Peptide It is extracted by a method comprising mixing trifluoroacetic acid boiled supernatants, except normal and normal state of the living body state, test method for assessing determine the progress of a disease state or condition. A test method for evaluating and discriminating a normal state and a non-normal state of a living body, a disease state, or a degree of progression of a disease using at least one of a protein, a digestion degradation product protein of the protein, and a digestion degradation product peptide of the protein as a marker a is, said protein is present as an intact protein in vivo in a normal state, which is digested decomposed into at least one digestible breakdown products proteins or peptides in a biological state other than the normal, the serum intact protein, the digestive breakdown products protein and / or type of the digestive breakdown products peptide, to measure the abundance and / or abundance, saw including obtaining a protein / partial peptide profile, wherein the protein to be measured, Digestion degradation product protein and / or Others are extracted by a method comprising digesting decomposition product peptides is mixed with trifluoroacetic acid boiling serum, a state other than the normal and the normal state of a living body of claim 1, wherein assessing the progression of the disease state or condition, inspection method to determine.   The test method according to claim 1 or 2, wherein two or more kinds of intact proteins, partial proteins, partial peptides, digestion degradation product proteins and / or digestion degradation product peptides are used as markers. The test method according to claim 2 or 3 , wherein the digestion degradation product protein or digestion degradation product peptide is a digestion degradation product by the action of a protease of an intact protein in a living body in a normal state. The test method according to claim 1 or 3 , wherein the partial protein or partial peptide is a partial expression product of an intact protein in a normal living body. The test method according to any one of claims 1 to 5 , wherein the state other than normal is inflammation, precancerous lesion, cancer or advanced cancer. The inspection method according to any one of claims 1 to 6 , wherein a normal state, chronic hepatitis, cirrhosis and liver cancer can be evaluated and discriminated. The inspection method according to any one of claims 1 to 7 , wherein the measurement is performed by an enzyme or fluorescent labeled antibody method or mass spectrometry.

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