JP2006300758A - Method for quantitatively determining target protein contained in living body origin sample by using internal standard peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel method for quantitatively determining a protein, which detects, identifies and quantitatively determines a target protein contained in a living body origin sample being a mixture of a plurality of proteins. <P>SOLUTION: A peptide corresponding to the target protein is synthesized and labeled with a stable isotope, and then its known amount is added to the living body origin sample as an internal standard peptide. Then, a protease is used to digest the protein into a peptide, and the peptide is separated by using a liquid chromatography and analyzed by a mass spectrometer. A signal square measure of the peptide derived from the protein is compared with that of the known amount of internal standard peptide, thereby quantitatively determining the target protein. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a method for identifying and quantifying proteins. More specifically, the present invention relates to a method for identifying and quantifying an arbitrary target protein from a biological sample that is a mixture of many kinds of proteins.

With the completion of the human genome analysis, a vast amount of genetic information was revealed. Analyzing this genetic information, understanding what kind of gene is working when, where and how much, and describing life activity is an issue. However, proteins are actually responsible for life activities, and the amount and amount of gene and protein working do not always match, so both genes and proteins are temporally, spatially and quantitatively It is thought that life activities can be understood only by obtaining information. Therefore, there is a need for a universal protein quantification method that can be used regardless of temporal and spatial differences.

As a method for specifically identifying and quantifying an arbitrary target protein from a mixture containing a plurality of proteins, an ELISA method or an RIA method is generally used. Since these methods use an antibody against the target protein, high-sensitivity detection is possible if a good-quality antibody is used. In addition, the use of multi-plates such as 96 wells is superior in that many types of samples can be analyzed at once. However, an antibody is indispensable for analysis, and it takes time to produce the antibody. Further, since the accuracy of measurement changes depending on the degree of specificity of the antibody used, there are problems that false positives are likely to appear. In the method using an antibody, only a target protein is identified by distinguishing one amino acid difference from a protein group having very similar amino acid sequences such as a modified specific protein, isozyme, isoform, protein derived from a single nucleotide polymorphism gene, etc. Is extremely difficult to identify and quantify.

The activity measurement method is a method of performing quantification by directly or indirectly detecting a product or by-product generated as a result of a chemical reaction catalyzed by the target protein when the target protein has enzyme activity. Although it is excellent in that the target protein can be easily detected and quantified, there is a problem that versatility is low because analysis is possible only with a protein having enzyme activity. In addition, since the measurement value varies depending on the measurement conditions such as the presence or absence of a substance that affects the activity, the measurement result does not necessarily accurately reflect the abundance in the living body.

Regarding the protein quantification method using stable isotopes, a method developed by Abersold et al. In the United States has been published ((Publication Number) Special Table 2002-523058). In this method, a cysteine residue contained in a protein is labeled with a biotinylation-modifying reagent, purified using the affinity for the labeled biotin, and individual proteins contained between two types of biological samples are obtained. It is a method of comparing quantities.
In this method, two types of labeling reagents having different masses incorporating stable isotopes are used to distinguish two types of proteins derived from biological samples by mass difference. It is very good in that it can easily compare multiple biological samples, but measurement errors are likely to occur due to the difference in labeling efficiency of the two types of labeling reagents, and the concentration of a specific protein cannot be quantified. There are drawbacks.

The method of Stott et al. In the United States has been reported as a method for quantifying protein concentration using stable isotopes (Rapid Commun. Mass Spectrom. 2004; 18: 491-498). In this method, the protein concentration can be quantified by using a peptide incorporating a stable isotope as a standard substance. This method is superior in that a calibration curve is prepared using a standard substance and the concentration is measured, but the target protein or target peptide can be obtained simply by separating all the peptides in the fragmented sample by reverse phase chromatography. Since the separation / concentration of the protein is insufficient, the signal pattern obtained is complicated, and it is difficult to quantify the trace amount of protein in the complex sample.

In view of the problems of the conventional protein quantification techniques as described above, the object of the present invention is to determine the abundance (concentration) of a target protein contained in a biological sample, its type, nature, location, characteristics of recognized antibodies, etc. Regardless, it is to provide a universal method that can be quantified.
More specifically, an object of the present invention is to provide a protein quantification method capable of realizing high-sensitivity detection, high-precision identification, and high-precision quantification without using an antibody or protein-specific activity.

The present inventors have focused on a protein quantification method characterized by quantifying a target peptide obtained by digestion with a mass spectrometer, instead of directly quantifying the target protein, as a method for solving the above-mentioned problems. As a result of intensive studies, we have found a stable isotope-labeled internal standard peptide design method and peptide separation / concentration method that enable high-sensitivity detection and high-precision identification, as well as peptide fragments for realizing high-precision quantification The present inventors have completed the present invention by finding a method for correcting the conversion processing efficiency.
That is, the present invention
(1) A method for identifying and quantifying a target protein in a biological sample containing a plurality of proteins using an internal standard peptide or internal standard peptide precursor, comprising the following (a) to (e) A method for identifying and quantifying a target protein characterized by having each step.
(a) a specific amino acid sequence having at least one amino acid sequence that has the same amino acid sequence as the target peptide obtained by subjecting the target protein to a predetermined fragmentation treatment and is labeled with a stable isotope; Internal standard peptide with mass difference, or
One or more amino acids having an amino acid sequence containing the same amino acid sequence as the target peptide obtained by subjecting the target protein to a predetermined fragmentation treatment and labeled with a stable isotope within the same amino acid sequence A step of preparing an internal standard peptide precursor having a specific mass difference by including
(b) A step of adding a known amount of an internal standard peptide or / and an internal standard peptide precursor to a biological sample and mixing them uniformly.
(c) The protein and the internal standard peptide or / and the internal standard peptide precursor contained in the biological sample are modified with an affinity tag labeling reagent, followed by the predetermined fragmentation treatment in the above step (a), and then the affinity Separating and concentrating tag-tagged target peptide and internal standard peptide using their affinity
(d) Analyzing the peptide of the separated / concentrated fraction with a mass spectrometer, and using the fact that the target peptide and the internal standard peptide are detected as a signal pair showing a specific different mass difference, a signal derived from the target peptide Identifying process
(e) The detected target peptide is subjected to internal sequence analysis by MS / MS using a mass spectrometer to identify the target protein, and the signal area or height of the target peptide and the known amount of the internal standard peptide. Step of quantifying target protein in biological sample by comparing signal area or height respectively (2) Using two or more kinds of internal standard peptides or internal standard peptide precursors with different masses for each target peptide The method for identifying and quantifying a target protein according to (1) above, wherein
(3) The internal standard peptide or internal standard peptide precursor subjected to the same modification as that in the target protein subjected to post-translational modification is used as the internal standard peptide or internal standard peptide precursor (1) Or the identification / quantification method of the target protein as described in (2).
(4) Identification of the target protein according to any one of (1) to (3) above, wherein a plurality of internal standard peptides or / and internal standard peptide precursors respectively corresponding to the plurality of target proteins are used. Quantitative method.
(5) The target according to any one of (1) to (4) above, wherein the internal standard peptide or the internal standard peptide precursor satisfies at least one of the following conditions (a) to (e): Protein identification and quantification.
(A) It does not contain an amino acid that undergoes non-physiological modification, such as a methionine residue that undergoes oxidation. (B) It does not have a glutamine or glutamic acid residue that causes cyclization at the N-terminus (c). For example, it contains at least one amino acid residue such as cysteine or lysine to be reacted with the affinity tag labeling reagent. (D) When fragmenting a peptide fragment, the fragmentation efficiency is high and no fragmentation error is included (e ) It relates to the mass being appropriate for the mass spectrometer used for the analysis.

According to the method of the present invention configured as described above, first, instead of directly quantifying proteins, peptide fragments obtained by digestion are quantified, so that they are not affected by the solubility of protein molecules, Proteins with various chemical properties can be handled in the same way. Second, by performing the modification using an affinity tag labeling reagent, the affinity tag labeled peptide can be easily separated and concentrated using affinity chromatography. Thirdly, according to the method of the present invention, since measurement is performed using a mass spectrometer, it is possible to quantify several fmol of protein, and by performing identification by peptide internal sequence analysis, false positive The target protein can be identified and quantified by discriminating one amino acid difference from a protein group having very similar amino acid sequences such as isozymes and isoforms. Fourth, by simultaneously using internal standard peptides corresponding to a plurality of target proteins, it is possible to quantify a plurality of target proteins from a single analysis. Fifth, a standard curve can be created for more accurate quantification by simultaneously using a plurality of types of internal standard peptides with a difference in mass for one type of peptide. Sixth, a fragment from which the peptide is cut out from the target protein by using a peptide (internal standard peptide precursor) to which the sequence to be removed upon digestion is added to the N-terminal and C-terminal of the internal standard peptide. High-accuracy quantification that corrects for errors due to the efficiency of the chemical reaction is possible. For example, when trypsin is used, fragmentation is suppressed when adjacent to a site where aspartic acid and glutamic acid having negative charges are cleaved. When fragmentation is suppressed in this way, more accurate quantification is possible by correcting the efficiency of the fragmentation reaction.

In the protein quantification method of the invention of the present application, first, a known amount of a stable isotope-labeled peptide is added as an internal standard to a protein solution extracted from a biological sample. By stable isotope labeling, a protein-derived peptide contained in a biological sample and a peptide added as an internal standard can be distinguished by a mass difference. Here, the stable isotope labeling of a peptide can be performed by incorporating a stable isotope during peptide synthesis. It is also possible to carry out stable isotope labeling by reacting a reagent containing a stable isotope. In this case, a reagent having a different isotope composition is used for the protein contained in the biological sample. Need to be labeled. The peptide added as an internal standard is a sequence that is unlikely to cause a change in mass due to artificial modification represented by oxidation or cyclization, and a sequence with high digestion efficiency when digesting into a peptide is selected. Furthermore, when using a length suitable for mass spectrometry, specifically a time-of-flight mass separator (TOFMS), an array in the range of 600 Da to 4000 Da is selected. In order to distinguish the family and isoform to which the protein belongs, it is necessary to select a specific sequence that is not conserved between the family and isoform. The protein is then digested into peptide fragments. As a method for digesting a protein into peptide fragments, a method using a proteolytic enzyme typified by trypsin and a chemical cleavage method typified by cyanogen bromide can be used.

The target peptide is separated from the mixture obtained by digesting the protein into peptide fragments. Separation is performed using liquid chromatography. Specifically, the separation can be carried out by combining cation exchange chromatography that performs separation utilizing the difference in the charge of the peptide and reverse phase chromatography that performs separation utilizing the difference in the hydrophobicity of the peptide. Biotin is avidin, maltose is maltose-binding protein, and glutathione is glutathione transferase, such as glutathione transferase. You can use graphy. Further, anion exchange chromatography, normal phase chromatography, and gel filtration chromatography can be used. By separating the target peptide from a mixture containing a plurality of types of peptides, detection sensitivity is improved, and accurate quantification is possible. Here, the separation of the target peptide is intended to increase the purity of the target peptide, and it is not necessary to exclude all other peptides.

The separated peptide is then detected using a mass spectrometer. By using a mass spectrometer, a target protein of several fmol can be detected. In addition, by performing MS / MS analysis, it is possible to analyze the internal sequence information of the peptide and to identify without false positives. The ionization method in mass spectrometry can be appropriately selected according to each apparatus. For example, electron ionization method (EI method), chemical ionization method (CI method), fast atom bombardment method (FAB method), electrospray ionization method (ESI method), atmospheric pressure chemical ionization method (APCI method), matrix-assisted laser desorption Various methods such as a deionization method (MALDI method) can be mentioned. In mass spectrometry, peptide-derived ions generated by various ionization methods are separated according to mass by an analyzer. For example, magnetic mass separator (Sector MS), quadrupole mass separator (QMS), time-of-flight mass separator (TOFMS), Fourier transform ion cyclotron mass separator (FT-ICRMS) Furthermore, an analyzer combining these is exemplified. Although it is possible to identify the peptide significantly from the elution position and mass of the chromatography, it is desirable to perform the internal sequence analysis of the detected peptide and perform the protein identification by performing MS / MS analysis.

By mass spectrometry, a signal (a) of an unknown amount of the target protein-derived peptide and a signal (b) of a known amount of the internal standard peptide are detected. The relationship between the area of each signal (m1, m2) and the amount of peptide contained in the measurement sample (x1, x2) is proportional.
m1: x1 = m2: x2
Indicated by
x1 = m1 × x2 / m2
Is established.
Therefore, since m1 and m2 are measured values and x2 is a known amount, the amount (x1) of the target protein can be measured.

When using multiple types of internal standard peptides with a mass difference of +9 Da, +15 Da, +21 Da, etc. for one type of peptide at the same time, the area (M9, M15, M21) of each internal standard peptide and the target From the relationship with the area (M0) of the protein-derived peptide, a calibration curve can be created and quantified.

The invention of the present application is a method for measuring the amount of protein. An internal standard peptide corresponding to a portion that undergoes post-translational modification such as phosphorylation, glycosylation, nitration, citrullination, etc. By synthesizing and using the peptide as an included peptide, it becomes possible to measure the amount of the target protein that has undergone post-translational modification. In addition, if an internal standard peptide for a peptide existing in a living body is synthesized and used, the amount of the peptide can be measured.

It is also possible to add each internal standard peptide for a plurality of target proteins to a biological sample. In this case, it is possible to simultaneously measure the amounts of a plurality of target proteins present in one sample.

Quantification of bovine serum albumin (BSA) In order to quantify bovine serum albumin (BSA), which is an internal standard peptide target protein, the following sequence was selected as an internal standard peptide from the amino acid sequence of BSA.
Internal standard peptide
BSA-3 (+ 9): SL * HTL * FGDEL ** CK
BSA-3 (+15): SL * HTL ** FGDEL ** CK
BSA-3 (+21): SL ** HTL ** FGDEL ** CK
L *: 1 Da heavy leucine
L **: 7 Da heavy leucine The above peptides were selected as peptides satisfying the following five conditions from the signals obtained by digesting BSA into peptides with trypsin and performing mass spectrometry.
1. Does not contain amino acids that undergo non-physiological modifications, such as methionine residues that undergo oxidation.
2. It does not have a glutamine or glutamic acid residue that causes cyclization at the N-terminus.
3. It contains one or more amino acid residues such as cysteine and lysine that are reacted with an affinity tag labeling reagent. Preferably, two or more amino acids to be reacted are not contained.
4. Fragmentation efficiency is high when peptide fragments are fragmented, and no fragmentation errors are included.
5. The mass is appropriate for the mass spectrometer used for the analysis. For example, when using a time-of-flight mass separator (TOFMS), it is in the range of 600 Da to 4000 Da.

Affinity tag labeling / peptide fragmentation In this example, the following known amounts of internal standard peptides were added to the proteins before affinity tag labeling.
BSA-3 (+9) 25 pmol
BSA-3 (+15) 50 pmol
BSA-3 (+21) 100 pmol
Affinity tag labeling of cysteine residues was performed using N-biotinoyl-N′-iodoacetylethylenediamine (hereinafter referred to as biotin) as an affinity tag label. By labeling with biotin, the mass of the peptide is increased by 326.42 Da.
Peptide fragments were prepared by adding trypsin to the labeled protein and the internal standard peptide.

Separation of peptides Peptides were separated by a combination of cation exchange chromatography, affinity chromatography and reverse phase chromatography.
Cation exchange chromatography is performed by connecting a polysulfoethyl A column (5 μm, 300 mm bead, 4.6 × 100 mm: PolyLC) to a high-performance liquid chromatography system (Vision Workstation: Applied Biosystems). I did it. The peptide mixture was adsorbed onto the column carrier using a phosphate buffer (pH 3.0) containing acetonitrile. After washing the column, the peptide was eluted by increasing the salt concentration.
Affinity chromatography was performed using the affinity of biotin used as an affinity tag labeling reagent for avidin. Specifically, an iCat cartridge-avidin column (4.0 × 15 mm: Applied Biosystems) was connected to a high performance liquid chromatography system (Vision Workstation: Applied Biosystems). Using a phosphate buffer (pH 7.2), the peptide mixture was packed into a column and adsorbed onto a column carrier. After washing the column, the peptide was eluted with 30% acetonitrile aqueous solution containing 0.4% trifluoroacetic acid.
Reversed phase chromatography was performed by connecting Symmetry C18 (3.5 μm, 1.0 × 150 mm: Waters) to a high performance liquid chromatography system (Aliance 2690: Waters). The peptide mixture was adsorbed onto the column carrier using an aqueous solution containing trifluoroacetic acid, and then the acetonitrile concentration was increased to elute the peptide.
In this example, the elution position in cation exchange chromatography and reversed-phase chromatography was examined by a preliminary experiment using the internal standard peptide alone, and then the experiment with this sample was performed. By conducting this preliminary experiment, it is possible to advance the experiment efficiently.

Peptides separated by liquid chromatography using a mass spectrometer were quantified using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (Applied Biosystems 4700 Proteomics Analyzer: Applied Biosystems). Among the obtained signals, four types of signal groups including three types of internal standard peptides were detected (FIG. 1). A calibration curve was created from the ratio of the added amount of three types of internal standard peptides (b, c, d) and the signal area (FIG. 2), and BSA was quantified. The quantitative results are shown in Table 1.

表１から、50 pmol のBSAを定量した結果、52.02 ± 1.69 pmol（n=3）であることが明らかとなった。この結果から、誤差4%未満で定量を行なえることが確認された。
このように、1つのペプチドに対して質量差をつけた複数の内部標準ペプチドを使用することで、検量線を用いた高精度の定量を行なうことが可能となる。

From Table 1, as a result of quantifying 50 pmol of BSA, it was revealed that it was 52.02 ± 1.69 pmol (n = 3). From this result, it was confirmed that quantification can be performed with an error of less than 4%.
As described above, by using a plurality of internal standard peptides having a mass difference with respect to one peptide, it is possible to perform highly accurate quantification using a calibration curve.

It is the figure which showed the example which used the some internal standard peptide which gave the mass difference with respect to one peptide demonstrated in the Example. (A) 50 pmol BSA-derived signal, (b) 25 pmol +9 Da internal standard peptide-derived signal, (c) 50 pmol +15 Da internal standard peptide-derived signal, (d) 100 pmol Shown are signals derived from the +21 Da internal standard peptide. FIG. 2 is a diagram in which a calibration curve is created from the addition amount and signal area ratio of the three types of internal standard peptides (b, c, d) shown in FIG. 1. The value of the amount of BSA-derived peptide is indicated by an arrow.

Claims (5)

A method for identifying and quantifying a target protein in a biological sample containing a plurality of proteins using an internal standard peptide or internal standard peptide precursor, comprising the following steps (a) to (e): A method for identifying and quantifying a target protein, comprising:
(a) a specific amino acid sequence having at least one amino acid sequence that is identical to the target peptide obtained by subjecting the target protein to a predetermined fragmentation treatment and labeled with a stable isotope; Internal standard peptide with mass difference, or
One or more amino acids having an amino acid sequence containing the same amino acid sequence as the target peptide obtained by subjecting the target protein to a predetermined fragmentation treatment and labeled with a stable isotope within the same amino acid sequence A step of preparing an internal standard peptide precursor having a specific mass difference by including
(b) A step of adding a known amount of internal standard peptide or / and internal standard peptide precursor to a biological sample and mixing them uniformly.
(c) The protein and the internal standard peptide or / and the internal standard peptide precursor contained in the biological sample are modified with an affinity tag labeling reagent, followed by the predetermined fragmentation treatment in the above step (a), and then the affinity Separating and concentrating tag-tagged target peptide and internal standard peptide using their affinity
(d) Analyzing the separated and concentrated peptides with a mass spectrometer, and detecting that the target peptide and internal standard peptide are detected as a pair of signals showing specific different mass differences. Identifying process
(e) The detected target peptide is subjected to internal sequence analysis by MS / MS using a mass spectrometer to identify the target protein, and the signal area or height of the target peptide and the known amount of the internal standard peptide. Quantifying target protein in biological samples by comparing signal area or height The method for identifying and quantifying a target protein according to claim 1, wherein two or more types of internal standard peptides or internal standard peptide precursors each having different masses are used for one target peptide. The internal standard peptide or internal standard peptide precursor subjected to the same modification as that in the target protein subjected to post-translational modification is used as the internal standard peptide or the internal standard peptide precursor. Identification and quantification of target proteins. The method for identifying and quantifying a target protein according to any one of claims 1 to 3, wherein a plurality of internal standard peptides or / and internal standard peptide precursors respectively corresponding to a plurality of target proteins are used. The method for identifying and quantifying a target protein according to any one of claims 1 to 4, wherein the internal standard peptide or the internal standard peptide precursor satisfies at least one of the following conditions (1) to (5).
(1) Does not contain amino acids that undergo non-physiological modifications, such as methionine residues that undergo oxidation (2) Does not have glutamine or glutamate residues that cause cyclization at the N-terminus (3) It contains at least one amino acid residue such as cysteine or lysine to be reacted with the affinity tag labeling reagent. (4) Fragmentation efficiency is high when fragmenting into peptide fragments (5) ) Appropriate mass for the mass spectrometer used for analysis

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