KR20110121842A - A mass spectrometric method of peptide quantitation by labeling peptides with n-methylpiperazine acetic acid and its isotopologues - Google Patents

Abstract

PURPOSE: A peptide quantitation using isotopologues of N-methylpiperazine acetic acid and a mass spectrometer is provided to purify and quantitate peptides with high reliability. CONSTITUTION: A peptide quantitation using N-methyl piperazine acetic acid substitution comprises: a step of treating a test sample with protease to cleave with peptide; a step of preparing isotopologue compounds in which entire or partial C, N, O, or H is substituted with ^13C, ^15N, ^18O or ^2H; a step of treating the cleaved peptide samples with isotopologue compounds for labeling; a step of mixing the labeled peptide samples; a step of scanning the samples using a mass spectrometer; and a step of analyzing MS/MS spectrum. The protease is trypsin.

Description

A mass spectrometric method of peptide quantitation by labeling peptides with N-methylpiperazine acetic acid and its isotopologues}

The present invention relates to a method for relative quantification of peptide complexes using isopologues and mass spectrometers of N-methylpiperazine acetic acid.

"Protein quantitation" is as important as "protein analysis" to identify the profile of a protein present in a sample. When a particular treatment is performed on a sample, the result is a change in protein profile and a quantitative change in individual proteins, so when comparing normal cells to cancer cells, any particular protein that can be a cancer cell marker can be applied to both normal and cancer cells. There may be quantitative differences, but in this case, quantitative analysis of the protein will make it easier to identify the proteins that differ. In the past, two-dimensional electrophoresis or 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) method was used to quantify these proteins, but recently, isotopes, which are compounds that are substituted for isotopes without two-dimensional electrophoresis, are used. After performing label substitution using (isotopologue), the mass spectrometry is widely used.

This labeling quantitative method can label each of the peptide complexes to be compared with different isotopes of different masses, mix them, and then perform one mass spectrometry to identify and quantify the individual peptides simultaneously. Because of the different masses and the same chemical properties of the same isotopes, the same peptides labeled with different masses behave identically in different separations and purifications, resulting in the same ionization, and only in the last spectrum. Representative isoisomeric compounds currently widely used include Isotopic Coded Affinity Tag (ITA), Stable Isopope Labeling with Amino Acid in Cell culture (SILAC), and isobaric Tags for Relative and Absolute Quantitation (iTRAQ).

ICAT specifically reacts with the -SH (thiol) group. Since only amino acids with Thiol are cysteine, ICAT is only labeled with peptides containing cysteine. This ICAT is a part of the iodoacetamide that clings to Thiol, an isotope coded tag that introduces isotopes, and biotin, which allows affinity chromatography to separate peptides bound to ICAT. It is composed of parts. When the ICAT reagent is reacted with the protein, the label is attached to the cysteine amino acid, the protein is cleaved with an enzyme, and the peptide with the ICAT label is selected by avidin affinity chromatography and analyzed by liquid chromatography-mass spectrometry. The two samples to be quantified relative are labeled with the same, but different, isotopic ICAT compounds. The two isotopes are structurally and chemically identical, but the nine carbons in the isotope coded tag are one of ¹² C and the other heavy ¹³ C. Thus, the same peptides with each isotope are distinguished and can be quantified in mass spectrometry (Gygi SP et al., Nat . Biotechnol ., 17: 994-999, 1999). However, since ICAT selectively labels the rare amino acid cysteine, the number of peptides that can be obtained from one protein is not so high that it is difficult to quantitatively reliably analyze proteins present in low concentrations. There are disadvantages such as the case where the tandem mass spectrometry spectrum does not improve.

SILAC was developed to quantify cell samples that can be grown in the laboratory. One cell line is grown in a medium containing common amino acids, and the other cell lines (cell types of different kinds or different treatment conditions) to be compared are grown in an isotopically labeled amino acid medium. For example, when making a medium, one is a normal lysine and the other is a heavy lysine with 6 carbons changed to ¹³ C. When the cells are grown, the cells grow and the lysine enters the protein. Subsequently, proteins obtained from cell samples grown in different mediums were mixed, cut into peptides, and subjected to liquid chromatography-mass spectrometry. Eventually, proteins from lysine containing heavy lysine were cut out of the peptides. Peptides are always 6 Da heavier than peptides from relative proteins (Ong SE et al., Mol . Cell . Proteomics , 1: 376-386, 2002). The principle of labeling amino acids of different sizes is the same as the method of ICAT. In ICAT, isotopic labeling is applied to proteins by protein reaction, whereas in SILAC, isotopically labeled amino acids are inserted into proteins with cell growth. Although SILAC does not chemically substitute isotopes for peptides, it does not only have to grow cells for a long time, but also cannot be used for samples such as human tissues.

iTRAQ quantitation is an amine-specific chemical tagging method that first hydrolyzes proteins with enzymes and then binds isobaric tags to all N-terminal and lysine amino acid peptides compared to ICAT. Quantitative and qualitative analysis of many proteins is possible. The tag used in the iTRAQ quantification method is divided into three parts: the N-hydroxysuccinimide group which reacts with the amino group of the N-terminus or lysine amino acid residue of the peptide, and the reporter group used for quantification ( reporter group), and a balance group for correcting the mass of the reporter group. ^{By using 13} C and ¹⁵ N isotopes appropriately, the mass of the reporter group varies from 114 to 117 Da for each tag, and the mass of the balance group varies from 31-28 Da to the contrary. Making the same isobaric chemistry identical, iTRAQ-labeled peptides will yield the same m / z of the same mass spectrometry scan, but when MS / MS fragmented in the mass spectrometer, the reporter would be 114, 115, 116 or The relative intensity of this peak is shown as 117 Da, indicating the relative amount of peptide in each sample, and the remaining fragmented ions in the MS / MS spectrum thus used are used to identify the peptide. This method not only has the advantage of testing the protein expression of 4, 6 or 8 different samples, including the reference sample, but because the isotopically substituted peptides have the same mass, Sensitivity is increased, making it the most widely used method today. However, since iTRAQ-labeled peptides cannot be quantified by MS1 alone, they can be quantified only through MS / MS fragmentation. Therefore, strong energy is required for MS / MS fragmentation. In the case of a mass spectrometer, its operation is difficult.

mTRAQ is a non-isobaric label that is labeled on the amine group of all peptides, unlike the creation of isotopically labeled heavy synthetic peptides for peptides representing specific proteins and the iTRAQ method using conventional reporter ions. Mass differences of 4-8 Da (each, Arg and Lys peptides) are generated, but the chemical and physically identical peptides are made, using a light reagent and a global internal standard (GIS) representing the mean value of the entire sample. And, using heavy reagents, multiple reaction monitoring (MRM) analysis with each GIS for each sample. Since GIS is used, it is easy to normalize various samples, and thus it is applicable to the validation studies of biomarker candidate proteins and peptides that require the use of many samples. However, since MTRAQ requires QTRAP or triple quadrupole MS with MRM, it is not applicable to general mass spectrometer.

Accordingly, the present inventors substituted two or three isotopes of the mTRAQ labeling compound N-methylpiperazine acetic acid to make two or three isotopologues, and applied them to a mass spectrometer to obtain peptides and It was confirmed that the protein can be identified and relative quantitative at the same time, and compared with the conventional ICAT method, it was found to be remarkably effective in quantifying protein complexes. In addition, this method was applied to colorectal cancer tissue samples to confirm that it is effective for the quantification of protein complexes, and the peptide quantification method using the en-methylpiperazine acetic acid label substitution of the present invention improves the reliability of protein identification and quantification compared to the conventional method. By confirming the elevation, the present invention was completed.

It is an object of the present invention to provide isotopic labeling substitution of N-methylpiperazine acetic acid and peptide quantification method using a mass spectrometer.

In order to achieve the above object, the present invention

1) treating the test sample with a protease and cleaving the peptide;

2) preparing an isopologue compound in which part or all of C, N, O or H is substituted with ¹³ C, ¹⁵ N, ¹⁸ O or ² H among the members of the compound represented by the following [Formula 1] Making;

In this case, R is a protecting group for protecting the carboxylic acid;

3) labeling the two or more peptide samples cleaved in step 1) by treating the different isotope compounds prepared in step 2), respectively;

4) mixing the labeled peptide sample;

5) scanning the mixed sample with MS mass spectrometry and MS / MS spectra using a mass spectrometer; And

6) Provides a method for quantifying peptides using en-methylpiperazine acetic acid label substitution comprising the step of identifying and quantifying peptides by analyzing MS / MS spectra.

Peptide quantification method using the isomer and mass spectrometer of the compound en-methylpiperazine acetic acid for peptide amino group substitution of the present invention is quantified using a large number of peptides compared to the ICAT method widely used to quantify protein complexes As well as giving higher quantitative values, the reliability of the identification is high due to the increase in type b ions due to N-methylpiperazine acetic acid. In addition, in the ion trap mass spectrometer, the identification rate for peptides having a large molecular weight (m / z> 1,500) can also be increased, so that peptides can be identified with higher reliability than conventional methods.

Figure 1 is a diagram showing the overall process of the present invention, showing an example of performing quantification in the MS spectrum, and identification in the MS / MS spectrum.
Figure 2 shows the results of liquid chromatography-mass spectrometry on plasma labeled and unlabeled plasma samples with N-methylpiperazine acetic acid:
A: Figure comparing the number of peptides and proteins identified; And
B: A graph showing Xcorr values of peptides commonly identified in each sample according to the amount of charge and amino acids at the terminal of the peptide.
FIG. 3 is a graph comparing the degree of matching between b-type and y-type ions of a peptide having increased Xcorr value and reduced peptide after substitution with N-methylpiperazine acetic acid label.
4 is a graphical representation of the same plasma sample labeled with light and heavy mTRAQ and then quantified by performing liquid chromatography-mass spectrometry to log ₂ .
FIG. 5 shows the results of liquid chromatography-mass spectrometry of colorectal cancer samples labeled with ICAT and N-methylpiperazine acetic acid:
A: Graph showing a comparison of the number of peptides and proteins identified; And
B: Graphical comparison of quantitative results of peptides and proteins identically identified in both experiments.
FIG. 6 is a graph showing Xcorr values of peptides commonly identified in liquid chromatography-mass spectrometry of colorectal cancer samples labeled with ICAT and N-methylpiperazine acetic acid according to charge amount and amino acid at the end of peptide.
Figure 7 shows the MS spectrum and MS / MS spectrum results of VGINYWLAHK (SEQ ID NO: 1) peptide of alpha-lactalbumin appearing at 12.61 minutes of elution time.
FIG. 8 is a diagram showing MS spectra and MS / MS spectra of the GEADAMSLDGGYLYIAGK (SEQ ID NO: 2) peptide of serotransferrin at 15.54 min.

Hereinafter, the present invention will be described in detail.

The present invention

1) treating the test sample with a protease and cleaving the peptide;

2) preparing an isopologue compound in which part or all of C, N, O or H is substituted with ¹³ C, ¹⁵ N, ¹⁸ O or ² H among the members of the compound represented by the following [Formula 1] Making;

[Formula 1]

,

,

In this case, R is a protecting group for protecting the carboxylic acid;

3) labeling the two or more peptide samples cleaved in step 1) by treating the different isotope compounds prepared in step 2), respectively;

4) mixing the labeled peptide sample;

5) scanning the mixed sample with MS mass spectrometry and MS / MS spectra using a mass spectrometer; And

6) Provides a method for quantifying peptides using en-methylpiperazine acetic acid label substitution comprising the step of identifying and quantifying peptides by analyzing MS / MS spectra.

The test sample of step 1) comprises cells, serum, plasma and blood, or proteins derived therefrom, proteins synthesized using some or all amino acid sequences of known proteins, and recombinant proteins prepared through genetic recombination techniques. It may be any one selected from the group, but is not limited thereto. The known protein may be synthesized using all or part of an amino acid sequence, extracted from a cell, produced from a microorganism using genetic recombination technology, or used in a biological sample such as blood, tissue, or cell, but is not limited thereto. .

The protease of step 1) may be any one or a combination of proteins extracted directly from various organisms, proteins prepared through genetic recombination technology, or any synthetic compound that degrades the protein, and is preferably trypsin. However, the present invention is not limited thereto.

N-methyl piperazine acetic acid (N-methyl piperazine acetic acid) of the step 2) is preferably substituted and labeled with the amino group of the N- terminal and lysine of the peptide, the R residue as a protecting group to protect the carboxylic acid , Methyl, benzyl, t-butyl, trimediline, dichlorophenol (2,3-dichlorophenol), tetrafluorophenol (2,3,5,6-tetrafluorophenol), tetrafluorosulfophenol (2,3,5 , 6-tetrafluoro-4-sulfophenol), N-hydroxysuccimide or N-hydroxysulfosuccinimide is preferably any one selected from the group consisting of, but not limited to And, more preferably, but is not limited to N-hydroxysuccimide (N-hydroxysuccimide).

Isotopologue of step 2) is a part or all of C, N, O, H in the element of N-methylpiperazine acetic acid is ¹³ C, ¹⁵ N, ¹⁸ O, ² Preferably substituted with H, but is not limited thereto.

The mass spectrometer of step 5) can be used as a general mass spectrometer, it is preferable to use MALDI or ESI, but is not limited thereto.

The analysis of step 6) preferably uses a commercially available software program or database, but is not limited thereto.

In a specific embodiment of the present invention, peptide identification is carried out using plasma proteins of humans to determine how the mass spectrometry of the peptide varies according to N-methylpiperazine acetic acid (mTRAQ) label substitution. As a result, 1,256 and 1,452 peptides were identified in samples labeled and unlabeled N-methylpiperazine acetic acid, respectively, of which 934 peptides were identified with or without N-methylpiperazine acetic acid labeled. It was confirmed that the same from the sample was identified (see A of Fig. 2). Collect MS / MS spectra for the same peptides up to the amount of charge among identically identified peptides, and compare the respective Xcorr values to compare the reliability of the tandem mass spectrometry according to the N-methylpiperazine acetic acid substitution. After substitution of the Razine acetic acid peptide, it was confirmed that the reliability of the tandem mass spectrometry spectrum was increased, which was due to the increase in type b ion fragment after labeling with N-methylpiperazine acetic acid (see FIG. 2B).

Proteins are identified by analyzing the amino acid sequence of peptides using tandem mass spectrometry, using an N-methylpiperazine acetic acid N-hydroxysuccimide ester compound. Substitution of methyl piperazine acetic acid (N-methylpiperazine acetic acid) is characterized in that the fragmentation pattern of the spectrum is improved. In general, fragmented peptides during Collision Induced Dissociation (CID-fragmentation of peptides due to collisions) have higher y-type daughter ions and relatively smaller amounts of b-type daughter ions (Yates JR et al., Anal. Chem., 70) . : 3557-3565. 1998). However, in the peptide substituted N-terminal and lysine residues, N-methylpiperazine acetic acid also increases the b-type ion, which is interpreted as an increase in the stability of the b-type daughter ion and the construction of a mobile proton model. can do. The N-terminus of the peptide cleaved with the protease is not high in basicity, and the b-type ion derived from the N-terminus is generally unstable compared to the y-type ion derived from the C-terminus. Because of this characteristic, the electrons move to the basic moiety and the y-type ions are relatively stabilized compared to the opposite b-type ions, so that the proton affinity increases when the basic N-methylpiperazine acetic acid is attached to the N-terminus, The b-type ion having the N-terminus becomes stable and the identification rate increases with the increase of the b-type ion.

In addition, in a specific embodiment of the present invention, after preparing a sample by treating the test sample with trypsin and cutting it with a peptide, half of the sample is reacted with a general en-methylpiperazine and labeled as light mTRAQ, and the other half is 3 It is called heavy mTRAQ after reacting with the isomer of N-methylpiperazine having 4 carbons of ¹³ C and one nitrogen of ¹⁵ N substituted with 4 Da larger than a general compound. After completion of the reaction, the samples were mixed with each other to obtain a mass spectrometry (MS spectrum) and a tandem mass spectrometry (MS / MS spectrum) by using a mass spectrometer equipped with reversed phase liquid chromatography (Thermo Scientific's LTQ XL-Orbitrap). The obtained MS / MS spectrum was analyzed using a database search program (TurboSequest version 27, revision 12), and the database was applied to human samples. The human international protein index database (IPI, versions 3.44, European Bioinformatics Institute, http: // Peptide quantitation was carried out using mTRAQ label substitutions in standard samples, plasma samples and colorectal cancer samples by analysis using www.ebi.ac.uk/IPI). Quantitative analysis of Std1 and Std2 samples confirmed that all six proteins were identified as at least 8 to 51 unique peptides, and all observed peptides were labeled at the N-terminal and lysine residues. It was confirmed that it was stuck. The quantitative results showed the same trends as the expected values of the standard samples. The difference between the observed and predicted values for the six types of proteins ranged from 4% to 27.6% and the CV values from 0% to 15.5%. As a result of human plasma sample analysis, 867 peptides with over 90% of peptide reliability could be identified, and when the graphs were replaced with log2 of light / heavy ratio of each peptide, a bell-shaped fraction with mean: standard deviation of -0.0009: 0.4537 was identified. The ratio of light / heavy was 0.9994 and 95% of the peptides were quantified with high reliability, ranging from 0.67 to 1.56.

In addition, 13,250 unique peptides were identified as a result of colorectal cancer tissue analysis, and the number of proteins was 2,418, and 59% of the 2,418 proteins were 1 to 4 unique peptides. 44% were identified as 1 to 4 spectra. In comparison with the ICAT method, it was confirmed that the mTRAQ label substitution can identify more peptides than the experiment through ICAT substitution, and the quantitative results of the peptides and proteins commonly identified in mTRAQ and ICAT are shown in log2. Even when the graph was replaced with a graph, it was found that the results of quantitative experiments using mTRAQ and ICAT were consistent with each other (see FIG. 5B).

As a result, the synergistic effect of the tandem mass spectrometry with mTRAQ labeling was reconfirmed in comparison with the ICAT experiment. Among the 947 peptides identified in mTRAQ and ICAT, 704 peptides with the same charge amount were identified. As a result, about 80% of peptides showed an increase in XCorr value in mTRAQ, confirming that more peptides ending in lysine were observed than those ending on arginine (see FIG. 6).

In addition, in a specific embodiment of the present invention, in order to confirm whether or not it is possible to simultaneously quantitate several peptide complexes, simultaneous quantitative analysis using three peptide complexes was carried out, and the N-methylpiperazine acetic acid label substitution was 2 In addition to the relative quantification of proteins and peptides included in the eggplant samples, it was confirmed that the relative quantification of proteins and peptides included in three or more samples was possible according to a combination of appropriate isopologues (see FIGS. 7 and 8).

Accordingly, N-methylpiperazineacetic acid and its isopologues can be usefully used to comparatively compare protein derivatives of various origins, and ICAT is well known as a method for quantifying protein bodies. Compared to the method, the protein identification and quantification reliability was improved, and thus it was confirmed that it was more effective in quantifying protein complexes.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

< Example  1> Test Sample Preparation

The test sample was dissolved in any sample containing the protein complex in a lysis buffer consisting of 7M urea, 2M thiourea, 4% CHAPS and 30 mM Tris. Precipitated with acetone. Protein precipitate was dissolved again in 50 mM Tris buffer (pH 8.0) containing 3M Urea, 50 mM tris (2-carboxyethyl phosphine) (TCEP) was added, and then at 60 ° C. for one hour. It was reduced, and 200 mM methyl methanethiosulfonate was added and reacted at 25 ° C. for 10 minutes. The reaction samples were diluted 10-fold by adding 50 mM Tris (pH 8.0) and treated with trypsin (Trypsin, purchased from Promega) at a ratio of 10: 1 (protein: trypsin) for 16 hours at 37 ^° C. . Thereafter, the sample was completed by removing the salt and dried with a C18 SPE cartridge.

< Example  2> yen Methylpiperazine  Acetic acid label substitution

The sample prepared in <Example 1> was dissolved in 500 mM triethyl ammonium bicarbonate, and methylpiperazine (methyl8.0) was added thereto and reacted at 25 ° C. for one hour. In this case, one of the two samples to be compared is reacted using a common N-methylpiperazine (N-methylpiperazine, pH 8.0) (hereinafter referred to as light mTRAQ), and the other one has three carbons of ¹³ C. Dogs were replaced with ¹⁵ N and isotopically reacted with an isopologue (hereinafter referred to as heavy mTRAQ) having 4 Da larger than the general compound.

< Example  3> Mass spectrometry with mass spectrometer

After the reaction, the samples are mixed with each other and a mass spectrometer equipped with reversed phase liquid chromatography (eg, Thermo Scientific's LTQ XL-Orbitrap) is used to perform mass spectrometry (MS spectrum) and tandem mass spectrometry (MS / MS spectrum). Got it. Specifically, liquid chromatography was used by flowing 10% to 40% of acetonitrile gradient in a column filled with C18 resin in a silica coated capillary. , Mass spectrometry, took a full-scan of the MS spectrum and performed MS / MS consecutively on five of the peaks of greatest intensity.

The MS / MS spectrum search was performed using a database search program (TurboSequest version 27, revision 12), which was applied to human samples. human international protein index database; IPI, versions 3.44, European Bioinformatics Institute, http://www.ebi.ac.uk/IPI).

< Experimental Example  1> yen Methylpiperazine  Reliability Improvement Effect of Peptide Tandem Mass Spectrometry by Acetic Acid Label Substitution

<1-1> mTRAQ  Peptide Identification by Label Substitution

Peptide identification was performed using human plasma proteins to determine how the mass spectrometry of peptides was changed by N-methylpiperazine acetic acid (mTRAQ) label substitution. Specifically, mTRAQ-labeled and unlabeled plasma samples were subjected to liquid chromatography-mass spectrometry for 40 minutes.

As a result, 1,256 and 1,452 peptides were identified from samples labeled and labeled, respectively. Of these, 934 peptides were identified identically from the two samples, with or without mTRAQ labeling (FIG. 2A).

<1-2> mTRAQ  Reliability Comparison of Tandem Mass Spectrometry with Substitution

To compare the reliability of tandem mass spectrometry according to mTRAQ substitutions, MS / MS spectra were collected for the same 625 peptides up to the charge amount among the 934 peptides identically identified to determine the reliability of each peptide in the Xcorr (SEQUEST search program). Indicating scale) values were compared. If one peptide had more than one MS / MS spectrum, a high XCorr value was chosen among them.

As a result, about 80% of the 625 peptides had increased XCorr values in mTRAQ-labeled samples, and an increase in XCorr values was observed primarily at +2 rather than charge amount +3, and more on peptides ending in lysine than peptides ending with arginine. Much has been observed (see B of FIG. 2). In general, peptides labeled with mTRAQ showed more b-type ions, whereas cID fragmented peptides had higher y-type daughter ions and relatively smaller amounts of b-type daughter ions (FIG. 3A).

<1-3> Xcorr  Correlation between value and b-type ion

In order to confirm whether the increase in the XCorr value mentioned above is due to this increased type b ion, the matching degree of b ion and y ion of the identified peptides was confirmed in all spectra, and for this, type b for each spectrum SEQUEST search was performed considering only ions or y-type ions.

As a result, for peptides with increased XCorr values, not only did the mTRAQ-labeled peptide show higher b ion matching than the peptide that was not, and the matching of y ions also increased in the mTRAQ-labeled peptide (FIG. 3). B).

Therefore, it was found that the reliability of the overall peptide tandem mass spectrometry was increased by increasing the b-type ion fragment after labeling with mTRAQ.

< Experimental Example  2> Quantitative Analysis of Standard Protein and Plasma Samples Using N-Methylpiperazine Acetic Acid Label Substitution

<2-1> Standard Protein Sample Analysis

To confirm the usefulness of N-methylpiperazine acetic acid in relative quantification through MS spectra of mass spectrometry, a standard protein mixture from bovine and human plasma samples were labeled with mTRAQ.

Specifically, standard protein samples include alpha-lactalbumin, beta-casein, serotransferrin, alpha-S1-casein and alpha obtained from cattle. -S-casein (alpha-S2-casein), pancreatic ribonuclease was prepared by mixing. Standard sample 1 (Std1) was prepared by mixing 10 ug, 10 ug, 20 ug, 25 ug, 25 ug and 10 ug in the above protein sequence, and standard sample 2 (Std2) was 10 ug, 20 ug, 10 ug, 5 Prepared by mixing ug, 5 ug and 50ug. Thereafter, according to the method of <Example 1> and <Example 2>, the standard sample 1 (Std 1) and the standard sample 2 (Std 2) are divided into two and made into a total of four samples, and then light or heavy Standard sample 1 (Std1L) labeled with light and labeled with mTRAQ, Standard sample 1 (Std1H) labeled with heavy, likewise prepared standard sample 2 (Std2L, Std2H) labeled with light and heavy, respectively, Mass spectrometry was performed in the same manner as in 3>.

As a result, two kinds of quantitative experiments (Std1L vs Std2H, Std2L vs Std1H) confirmed that six proteins were identified as at least 8 to 51 single peptides, and the N-terminus and lysine residues of all observed peptides were identified. It was confirmed that mTRAQ was labeled with the amino group. In addition, the quantitative results of the mTRAQ-labeled standard protein showed the same tendency as the expected value of the standard sample. The difference between the observed value and the expected value for the six kinds of proteins was 4% to 27.6%, and the CV value was 0. The percentage was 15.5% (Table 1).

sample Standard Sample 1 (Light)
vs Standard Sample 2 (Heavy) Standard Sample 1 (Heavy)
vs Standard Sample 2 (Light) protein real
(observed)
ratio
Mean ± Standard Deviation prediction
(expected)
ratio Difference
(error)
(%) real
(observed)
ratio
Mean ± Standard Deviation prediction
(expected)
ratio Difference
(error)
(%) Alpha-lactalbumin
(Alpha-lactalbumin) 0.96 ± 0.03 One -4 0.93 ± 0.06 One -7 Beta-casein
(Beta-casein) 0.43 ± 0.00 0.5 -14 1.56 ± 0.06 2 -22 Serotransferin
(Serotransferrin) 1.87 ± 0.13 2 -6.5 0.42 ± 0.04 0.5 -16 Alpha-S1-casein
(Alpha-S1-casein) 3.93 ± 0.37 5 -21.4 0.18 ± 0.01 0.2 -10 Alpha-S2-casein
(Alpha-S2-casein) 3.62 ± 0.23 5 -27.6 0.21 ± 0.02 0.2 5
Pancreatic ribonucleases
Ribouclease
pancreatic) 0.17 ± 0.02 0.2 -15 4.52 ± 0.7 5 -9.6

<2-2> Human Plasma Sample Analysis

Quantitative experiments were performed by full-scan after mTRAQ label substitution in human plasma samples. Specifically, the human plasma sample was removed by using a MARS column of Agilent's MAR 6 large amounts of protein and concentrated through ultrafiltration (ultrafiltration). The same sample was divided in half and one was prepared to label the light mTRAQ and the other to label the heavy mTRAQ. Subsequently, after removing the six relatively large amounts of protein from plasma components, one was labeled in half with light mTRAQ and the other was labeled with heavy mTRAQ, and then the two samples were mixed and subjected to liquid chromatography-mass spectrometry. .

As a result, 867 peptides with more than 90% reliability were identified, and the mean ± standard deviation was -0.0009 ± 0.4537 and showed a bell-shaped distribution when the quantitative results of each peptide were replaced by log2. The average is 0.9994, it can be seen that 95% of the peptides were quantified with high reliability, ranging from 0.67 to 1.56 (Fig. 4).

< Experimental Example  3> N- Using Colorectal Cancer Tissue Samples Methylpiperazine  Acetic acid label substitution method ICAT  Comparison of Quantitative Analysis

To compare and analyze the relative quantification by mTRAQ-labeled substitution and the relative quantification by ICAT, colorectal cancer and peripheral colon tissue extracts were labeled with mTRAQ and ICAT. Analysis spectra were compared.

Specifically, colorectal cancer tissue samples were used to receive the MSS type cancer tissue and the surrounding colon tissue matched to three patients. The obtained tissue was washed with physiological saline, dissolved using a lysis buffer consisting of 7M urea, 2M thiourea, 4% CHAPS, and 30 mM Tris, precipitated with acetone, and then 50 mM tris containing 3M Urea. After dissolving in buffer buffer (Tris buffer, pH 8.0), cancer tissues were labeled with heavy mTRAQ and surrounding colon tissues were labeled with light mTRAQ, and each sample was labeled with ICAT, followed by liquid chromatography-quantitative analysis. Was performed.

As a result, 13,250 single peptides were identified by mTRAQ label substitution method and 5,147 single peptides were identified by ICAT method, of which 947 were identified in common in both experiments. In addition, 2,418 and 1,955 proteins were identified in each experiment, and a total of 3,290 proteins were identified through the two experiments, of which 1,083 proteins were commonly identified in both experiments (A of FIG. 5).

However, 173 proteins identified as peptides with cystine in mTRAQ substitutions were not identified in ICAT, 18% of peptides identified in ICAT were commonly identified in mTRAQ, and 55% of proteins identified in ICAT experiments. It was also commonly identified in mTRAQ. In addition, in the case of mTRAQ substitution method, 519 (21%) proteins were identified through one peptide, and 455 (23%) proteins were identified by ICAT quantification method in common. In addition, 59% of the 2,418 proteins in the mTRAQ experiment were identified as 1 to 4 single peptides, 44% as 1 to 4 scans, whereas 72% were 1 in the ICAT assay. Were identified as four single peptides, and 65% were identified from 1 to 4 scans.

Therefore, it was confirmed that mTRAQ-labeled substitution could identify more peptides than experiments using ICAT substitution. When quantitative results of peptides and proteins commonly identified in mTRAQ and ICAT were substituted by log 2, a graph was drawn. , The correlation coefficient between peptides was 0.842, and the correlation coefficient between proteins was 0.707, which indicates that the results of quantitative experiments using mTRAQ and ICAT were in good agreement with each other (FIG. 5B).

As a result, the synergistic effect of the tandem mass spectrometry with mTRAQ labeling was reconfirmed in comparison with the ICAT experiment. Among the 947 peptides identified in mTRAQ and ICAT, 704 peptides with the same charge amount were identified. As a result, about 80% of peptides showed an increase in XCorr value in mTRAQ, confirming that more peptides ending in lysine were observed than peptides ending in arginine (FIG. 6).

< Experimental Example  4> Yen Methylpiperazine  Simultaneous Quantitative Analysis of Three Peptide Complexes by Acetic Acid Label Substitution

Accuracy of quantification by label substitution of N-methylpiperazine acetic acid in the relative quantification of two peptide complexes through MS spectra of mass spectrometry in <Example 2> and <Example 3> The superiority of the identification was confirmed, and simultaneous quantitative analysis using the three peptide complexes was performed to confirm whether or not relative quantification of several peptide complexes could be performed simultaneously by the same method.

Specifically, three standard composite samples were prepared, each cut with trypsin, and then light mTRAQ, heavy mTRAQ, and a third isotope in which two nitrogen and six carbon atoms were substituted with ¹⁵ N and ¹³ C, respectively ( Δ8 mTRAQ). Standard composite sample 1 is alpha-lactalbumin, beta-casein, ceserotransferrin, alpha-S1-casein, alpha-S2 -Casein (alpha-S2-casein) and pancreatic ribonuclease contained 1 mg, 5 mg, 10 mg, 0.5 mg, 0.5 mg and 3 mg, respectively, and were labeled with light mTRAQ. Standard complex sample 2 contained 5 mg, 10 mg, 1 mg, 0.5 mg, 0.5 mg and 3 mg of the protein in order, respectively, and was labeled with heavy mTRAQ. , 1 mg, 5 mg, 1.5 mg, 1.5 mg, 1 mg, and labeled with Δ8 mTRAQ. Thereafter, three kinds of labeled standard composite samples were mixed together, cut into trypsin, but plasma samples not labeled with N-methylpiperazine acetic acid were added to make the sample complex, and then liquid chromatography-mass spectrometry was performed.

As a result, a total of 779 peptide fragments were identified, including peptide fragments of standard protein and peptide fragments of plasma, 79 proteins were identified, and all six standard proteins were detected. Alpha-lactalbumin is 30 peptides, beta-casein is 3 peptides, serotransferrin is 46 peptides, alpha-S1-casein (alpha-S1) -casein was identified as 11 peptides, alpha-S2-casein was identified as 9 peptides, and pancreatic ribonuclease was identified as 8 peptides. Peptides VGINYWLAHK of alpha-lactalbumin and peptide GEADAMSLDGGYLYIAGK of serotransferrin are shown in FIGS. 7 and 8.

In addition, three pairs of peaks (m / z = 740.93, 744.94, 748.94) appearing at 12.61 minutes of elution time showed that almost all cleaved ions in the MS / MS spectrum matched the fragment theoretical mass of the VGINYWLAHK (SEQ ID NO: 1) peptide. In addition, it was found that the quantitative ratios 1: 5: 10 of standard samples 1, 2, and 3 were correctly implemented in the MS spectrum, as well as three pairs of peaks (m / z = 1056.04, 1060.04) at 15.54 min. , 1064.05) Almost all cleaved ions in the MS / MS spectrum fit the fragmented theoretical mass of the GEADAMSLDGGYLYIAGK (SEQ ID NO: 2) peptide, and the quantitative ratios of standard samples 1, 2, and 3 in the MS spectrum were 10: 1: 5 Was found to be properly implemented.

Thus, N-methylpiperazine acetic acid label substitutions are not only relative quantities of proteins and peptides contained in the two samples, but also relative amounts of proteins and peptides contained in three or more samples depending on the appropriate isopologue combination. It can be seen that.

As described above, the peptide quantification method using the isomer and mass spectrometer of the compound en-methyl piperazine acetic acid for peptide amino group substitution of the present invention can quantitate peptides with higher reliability than conventional quantification methods. It can be usefully used for quantifying peptides and proteins for disease biomarker development.

<110> Korea Institute of Science and Technology <120> A mass spectrometric method of peptide quantitation by labeling          peptides with N-methylpiperazine acetic acid and its isotopologue <130> 10p-03-17 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> alpha-lactalbumin peptide <400> 1 Val Gly Ile Asn Tyr Trp Leu Ala His Lys   1 5 10 <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> serotransferrin peptide <400> 2 Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Tyr Leu Tyr Ile Ala   1 5 10 15 Gly lys        

Claims (6)

1) treating the test sample with a protease and cleaving the peptide;
2) preparing an isopologue compound in which part or all of C, N, O or H is substituted with ¹³ C, ¹⁵ N, ¹⁸ O or ² H among the members of the compound represented by the following [Formula 1] Making;
[Formula 1]

,
In this case, R is a protecting group for protecting the carboxylic acid;
3) labeling the two or more peptide samples cleaved in step 1) by treating the different isotope compounds prepared in step 2), respectively;
4) mixing the labeled peptide sample;
5) scanning the mixed sample with MS mass spectrometry and MS / MS spectra using a mass spectrometer; And
6) Peptide quantification method using en-methylpiperazine acetic acid label substitution comprising analyzing MS / MS spectra to identify and quantify peptides.
The method of claim 1, wherein the protease of step 1) is trypsin.
The method of claim 1, wherein in the formula (1) of step 2) as the protecting group to protect the carboxylic acid, methyl, benzyl, t-butyl, trimediline, dichlorophenol (2,3-dichlorophenol), Tetrafluorophenol (2,3,5,6-tetrafluorophenol), tetrafluorosulfophenol (2,3,5,6-tetrafluoro-4-sulfophenol), N-hydroxysuccimide or ethylene Peptide quantification method, characterized in that any one selected from the group consisting of N-hydroxysulfosuccinimide.
The method of claim 3, wherein the R residue is N-hydroxysuccimide.
2. The method of claim 1, wherein the mass spectrometer of step 4) is MALDI or ESI.
The method of claim 1, wherein the analysis of step 6) uses a commercially available software program or database.

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