JP6365863B2 - Database creation method for peptide peak identification and quantification in mass spectrometry - Google Patents

Description

  The present invention relates to a peptide peak elution time for identifying and / or quantifying peptide peaks of peptides constituting a plurality of target proteins by analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS). And a method for creating a database storing MRM transitions and peak intensities corresponding to the elution time of the peptide peak, a database created by using such a method, and LC-MS / MS using such a database. The present invention relates to a method for identifying and quantifying peptide peaks of peptides constituting a target protein in a sample by analysis.

  Proteomics is a method for comprehensive analysis of proteins, and is an important method for life science, especially biomarker research. In recent years, proteomics is expected not only as a comprehensive identification of proteins present in samples, but also as a comprehensive quantitative comparison tool (quantitative proteomics) for comprehensive identification and quantification of proteins that vary between different samples. Various techniques have been developed and used so far (Non-Patent Document 1). Quantitative proteomics is a shotgun proteomics, that is, a method based on the method of proteolytic enzyme treatment of a sample containing a large number of proteins to separate peptide fragments and identify the peptide fragments using mass spectrometry. Proteins that can be produced are limited to those identified in the same analysis sample. Therefore, the number of proteins that can be comparatively analyzed has been increased by improving mass spectrometry or a method of separating peptide fragments before mass spectrometry.

  Proteomics using multiple reaction monitoring (MRM) is a proteomic that does not use shotgun proteomics. Proteomics using MRM is a technique for quantifying a target protein using a specific MRM transition based on the mass-to-charge ratio (m / z) of a precursor (precursor) ion (Q1) and a product ion (Q3) ( Non-patent documents 1 to 3) By using such a technique in combination with an internal standard peptide labeled with a stable isotope, quantitative changes in target proteins in different samples can be quantified with high sensitivity and high accuracy. (Non-patent documents 4 to 6, Patent document 1). However, it has been a problem that only the target protein can be quantified, and the number of target proteins that can be quantified is limited by the scanning speed in mass spectrometry and the number of labeled internal standard peptides.

  Recently, a SWATH-MS acquisition method has been developed as a new data acquisition method in LC-MS / MS (Non-Patent Documents 4 and 7). With this SWATH-MS acquisition method, it is possible to acquire all MS / MS data from a sample and perform MRM analysis using an MRM transition. Although the data acquired by SWATH includes information on proteins expressed at a very low level (Non-patent Document 4), the data acquired by SWATH is the same as the conventional mass spectrometry method such as shotgun proteomics. The expression level is determined by quantitative analysis (standard SWATH quantification method) based on information such as MRM transitions and elution times (also referred to as “retention times [RT]”) for the identified proteins. Proteins that were not identified in the same analysis sample due to low values cannot be quantified.

International Publication No. 2007/055116 Pamphlet

Malmstrom, L., et al. Proteomics 11, 2947-2956 (2011) Gillette, M.A. & Carr, S.A. Nat Methods 10, 28-34 (2013) Surinova, S., et al. J Proteome Res 10, 5-16 (2011) Gillet, L.C., et al. Mol Cell Proteomics 11, O111 016717 (2012). Kamiie, J., et al. Pharm Res 25, 1469-1483 (2008) Ohtsuki, S., et al. J Pharm Sci 100, 3547-3559 (2011) Liu, Y., et al. Proteomics 13, 1247-1256 (2013)

  An object of the present invention is to provide a method for creating a database for identifying and quantifying peptide peaks of peptides constituting a target protein with high sensitivity and comprehensively by analysis using LC-MS / MS, An object of the present invention is to provide a method capable of identifying and quantifying peptide peaks of a peptide that constitutes a target protein with high sensitivity and comprehensive analysis by using LC-MS / MS.

  The time until the peptide constituting the target protein is eluted from the LC-MS / MS liquid chromatograph and detected by the mass spectrometer, that is, the elution time (also referred to as retention times (RT)) is the peptide peak. In order to solve the above-mentioned problems, the present inventors have studied the peptides identified between the analysis samples. Based on the elution time of the common peptide peak group among the peak groups, the elution time of the peptide peak group between analysis samples was standardized, and a database in which the standardized elution time was accumulated was created. It was found that the elution time of more than one peptide peak can be accumulated with high accuracy. Analyzing the target protein in the sample using LC-MS / MS, identifying the peptide peak to be measured, and validating the peptide peak from the identified peptide peak, using the standard SWATH quantification method It was confirmed that the increase and decrease of peptide peaks that could not be identified could be detected with high accuracy, and the present invention has been completed based on these findings.

That is, the present invention provides (1) identification and / or quantification of peptide peaks of peptides constituting a plurality of target proteins by analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS). A method of creating a database storing peptide peptide elution times, MRM transitions corresponding to the peptide peak elution times, and peak intensities, comprising the following steps (a) to (f): On how to do.
(A) Obtain the standard elution time of the peptide peak group and the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak group obtained by mass spectrometry using LC-MS / MS, and store them in the database The step of:
(B) obtaining the elution time of the peptide peak 'group, and the MRM transition and peak intensity corresponding to the elution time of the peptide peak' group, obtained by mass spectrometry using LC-MS / MS;
(C) selecting a peptide peak common to the peptide peak ′ group from the peptide peak group as a peptide peak group for standardization;
(D) Based on the standard elution time of the peptide peak group for standardization selected in the step (c), the standard elution time of the peptide peak group and the elution time of the peptide peak 'group are aligned, and the peptide peak' Normalizing the elution time of the group to the standard elution time of the peptide peak group, and obtaining the standard elution time of the peptide peak 'group;
(E) The step of incorporating the standard elution time of the peptide peak 'group obtained in step (d) into the standard elution time of the peptide peak group and storing it in the database as the standard elution time of a new peptide peak group;
(F) storing the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak 'group in the database;

  Moreover, this invention relates to the method as described in said (1) characterized by repeating the process of (2) process (b)-(f) 2-25 times.

  The present invention also provides (3) a database for identifying and / or quantifying peptide peaks of peptides constituting a plurality of target proteins by analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS). An elution time of 100,000 or more peptide peaks prepared using the method described in (1) or (2) above, and an MRM transition and a peak intensity corresponding to the elution time of the peptide peak It relates to a database characterized by that.

In addition, the present invention provides (4) peptide peaks of peptides constituting one or more target proteins in one or more samples by analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS). The present invention relates to a method for identification, comprising the following steps (A) to (E).
(A) A step of preparing a target peptide group by subjecting the target protein group in the sample to a fragmentation treatment with a protein digestive enzyme;
(B) Using the target peptide group prepared in step (A), analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS) is performed, and the peptide peak elution time, mass-to-charge ratio (m / Z), and obtaining data including MRM chromatogram information;
(C) The target peptide peak group is identified based on the mass-to-charge ratio (m / z) of the peptide peak obtained in step (B), and described in the above (1) or (2) from the target peptide peak group A step of selecting a peptide peak common to the peptide peak group stored in the database created by using the method as a target peptide peak group for standardization;
(D) Based on the elution time of the target peptide peak group for standardization selected in step (C), a database created using the elution time of the target peptide peak group and the method described in (1) or (2) above The standard elution time of the peptide peak group stored in the database prepared using the method described in (1) or (2) above is aligned with the standard elution time of the peptide peak group stored in Shifting to the elution time of the peak group, and obtaining the shifted standard elution time and the MRM transition and peak intensity corresponding to the shifted standard elution time as a reconstructed ion library;
(E) Using the reconstructed ion library acquired in step (D), the MRM chromatogram of the peptide peak is extracted from the data including the MRM chromatogram information acquired in step (B), and the target peptide is extracted from the MRM chromatogram. Identifying a peak group;

Further, the present invention (5) further comprises the following steps (F) and (G) for quantifying the peptide peak of the peptide constituting the target protein among a plurality of samples ( It relates to the method described in 4).
(F) A step of selecting a peptide peak for quantification of the target peptide peak group from the target peptide peak group identified in step (E) based on the ratio of the peak area and the peak intensity;
(G) The peptide peak area for quantification of the target peptide peak group selected in the step (F) is calculated for a plurality of samples, and the peptide peak of the peptide constituting the target protein among the plurality of samples is calculated from the ratio of the calculated peak areas. Quantifying

  The present invention further relates to (6) the method according to (4) or (5) above, wherein the fragmentation treatment with a protein digestive enzyme is a fragmentation treatment with a combined use of trypsin and lysylendopeptidase.

  According to the present invention, peptide peaks of peptides constituting the target protein can be identified and quantified with high sensitivity and comprehensiveness, so that samples from healthy individuals and samples from patients with various diseases such as cancer, Alzheimer and heart disease, It is possible to identify and quantitate a protein of interest whose expression level fluctuates in a highly sensitive and comprehensive manner, and is useful in the biomarker search field and the disease diagnosis field using biomarkers.

1A and 1B show an outline of a database creation method of the present invention. 1C-E are diagrams showing the results of creating a database of the present invention using human liver microsome (HLM) fractions. FIG. 2A shows an outline of a method for reconstructing an ion library using the database of the present invention. FIG. 2B shows the difference between the measured value RT and the corrected (shifted) nRT when the ion library is reconstructed using the database of the present invention. FIG. 2C is a diagram showing the results of comparing the areas of peptide peaks identified and quantified using standard SWATH quantification methods in two experiments (“Exp1” and “Exp2” in the figure). FIG. 2D is a diagram showing the results of comparing the areas of peptide peaks identified and quantified using the database of the present invention by two experiments (“Exp1” and “Exp2” in the figure). FIG. 2E shows the results of comparing peptide peak areas extracted and validated from peptide peaks identified using the database of the present invention by two experiments (“Exp1” and “Exp2” in the figure). FIG. FIG. 2F shows the number of MRM transitions among the peptide peaks extracted by performing peptide peak validation from the peptide peaks identified using the database of the present invention by two experiments (“Exp1” and “Exp2” in the figure). FIG. 3 is a diagram showing the results of extracting three or more peptide peaks and comparing their areas. FIG. 3 is a diagram showing the results of SWATH quantitative analysis using the model sample according to the database of the present invention.

  The database of the present invention includes 10 for identifying and / or quantifying a plurality of peptide peaks of a peptide constituting a plurality of target proteins by analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS). The elution time of 10,000 or more peptide peaks, the MRM transition corresponding to the elution time of the peptide peak, and the peak intensity are stored. The “peptide peak” stored in the database of the present invention contains the target protein. Peaks from the constituent peptides are included. The database of the present invention can be created using a method comprising the following steps (a) to (f) (hereinafter sometimes referred to as “the present database creation method”).

Step (a)
In step (a), the standard elution time of the peptide peak group and the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak group obtained by mass spectrometry using LC-MS / MS are obtained, and the database is obtained. To store. The above "standard elution time of peptide peak group obtained by mass spectrometry using LC-MS / MS and MRM transition and peak intensity corresponding to the standard elution time of such peptide peak group" The obtained sample (target protein sample) was fragmented with a protein digestion enzyme, and after preparing a peptide group containing peptides constituting the target protein, the analysis was performed using LC-MS / MS. It may be the standard elution time of a peak derived from a peptide group (peptide peak group), or the MRM transition or peak intensity corresponding to the standard elution time of the peptide peak group, or obtained by a third party performing the above analysis. Corresponding to the standard elution time of the peptide peak group and the standard elution time of the peptide peak group. It may be a transition and the peak intensity (Data [Information of).

  The target protein sample is not particularly limited as long as it is a sample containing the target protein. Specifically, cells, tissues, blood and the like collected from a living body are freeze-thawed, French press, glass beads, homogenizer, Unfractionated sample obtained by crushing using an ultrasonic crusher etc., and cell membrane fraction obtained by fractionating such unfractionated sample by fractional centrifugation, sucrose density gradient centrifugation, etc. A sample and a cytoplasm fraction sample can be mentioned.

  The protein digestion enzyme is not particularly limited as long as it is an enzyme that can be digested (cut) at a specific amino acid site of a protein to prepare a fragmented peptide. Specifically, trypsin, chymotrypsin, endoproteinase Glu-C, Mention may be made of endoproteinase Lys-C, endoproteinase Arg-C, endoproteinase Asn-C, lysyl endopeptidase, and clostripain. The fragmentation treatment with a protein digestion enzyme is not particularly limited as long as it is a fragmentation treatment with one or a plurality of protein digestion enzymes, but from the viewpoint of efficient fragmentation treatment, a fragmentation treatment with a plurality of the above protein digestion enzymes is preferable. Specifically, a fragmentation treatment using a combination of trypsin and lysyl endopeptidase can be preferably exemplified.

  In the analysis using LC-MS / MS, peptide groups are first separated by LC (liquid chromatography) of LC-MS / MS. Here, as liquid chromatography, specifically, cation exchange chromatography that performs separation using the difference in the charge of the peptide or reverse phase chromatography that performs separation using the difference in the hydrophobicity of the peptide. It may be mentioned, and may be a combination of both.

  Next, tandem mass spectrometry (MS / MS) is performed on each separated peptide. The ionization method in mass spectrometry is preferably an electrospray ionization method (ESI method), which is a soft ionization method. In mass spectrometry, peptides ionized by various ionization methods are separated according to mass by an analyzer. Examples of the analyzer include a magnetic mass separator (Sector MS), a quadrupole mass separator (QMS), a time-of-flight mass separator (TOFMS), and a Fourier transform ion cyclotron mass separator (FT-ICRMS). Or a combination thereof.

  As a method from detection of ionized peptides to acquisition of MS / MS data, a method in an automated measurement mode is preferable. Specifically, an IDA (Information Dependent Acquisition) measurement method, a DDA (Data Dependent Acquisition) measurement method, etc. Can be mentioned. The obtained MS / MS data is imported into protein identification software such as Protein Pilot, Peak View, MASCOT, etc., and peptide groups are identified using protein databases such as UniProt, EMBL, Swiss-Prot. Some of the identified peptide groups have a peptide peak with low reliability, so those with a highly reliable peptide peak from among the peptide peaks of the identified peptide group (peptide peak group) As a highly reliable peptide peak, MS / MS data of the peptide group identified by the above software is converted into an ion library file (text file) and exported to Access (manufactured by Microsoft). Thus, it is possible to select based on a peptide confidence score (peptide confidence score) which is an index indicating peptide reliability (the reliability score when the reliability is 100% is shown as 1.00), for example, A peptide with a score greater than 0.85, a peptide greater than 0.90, a peptide greater than 0.92. Peptides greater than 0.94, peptides greater than 0.95, peptides greater than 0.96, peptides greater than 0.97, peptides greater than 0.98, peptides greater than 0.99, and the like. More than 99 peptides are more preferred.

For the peptide peak group, information on the elution time in liquid chromatography and the MRM transition and peak intensity corresponding to the elution time are acquired and stored in a database. In the present specification, the term "MRM transitions", ionized peptide having a particular mass mass filter (Q1) and a gas collision-induced cleavage to pass (precursor ions): caused by (CID C ollision I nduced D issociation ) the It means a combination of mass filters (Q3) that allow the precursor ions to be fragmented (product ions) (combination of mass-to-charge ratio [m / z] of Q1 and Q3). The peptide peak group is identified a plurality of times in the peptide-derived peak by the software, and as a result, a plurality of different elution times may be obtained even for the same peptide peak. In that case, an average elution time obtained by averaging the elution times of peptide peaks is obtained and stored in a database. Moreover, the peptide peak may be misidentified by the above software, and the elution time of the misidentified peptide peak is greatly different from the elution time of the positively identified peptide peak. For this reason, it is preferable to exclude erroneously identified peptide peak data, and examples of the method include a method of excluding by selecting the peptide peak elution time and the standard deviation (SD) of the average elution time. Examples of the standard deviation of the elution time include, for example, less than 1.0 minute, less than 0.5 minute, less than 0.4 minute, less than 0.3 minute, and less than 0.2 minute. Less than 2 minutes are preferred.

  The database preferably further stores UniProt accession numbers corresponding to the standard elution times of peptide peak groups, and related information such as amino acid sequences and valences of peptides including information on peptide modifications. With this information, the peptide peak can be further characterized and can be used to specify the amino acid sequence of the peptide corresponding to the peptide peak, ie, the peptide constituting the protein of interest.

Step (b)
In the step (b), the elution time of the peptide peak 'group, the MRM transition and the peak intensity corresponding to the elution time of the peptide peak' group obtained by mass spectrometry using LC-MS / MS are obtained. The above "elution time of peptide peak 'group obtained by mass spectrometry using LC-MS / MS, and MRM transition and peak intensity corresponding to the elution time of such peptide peak'group" actually includes the target protein. A sample containing '(target protein' sample) is fragmented with a protein digestion enzyme to prepare a group of peptides containing peptides that constitute the target protein ', and then analyzed using LC-MS / MS. In addition to the standard elution time of the peak derived from the peptide group (peptide peak group) and the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak group, the peptide obtained by the above analysis The standard elution time of the peak 'group, the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak' group (data [ Distribution]) is also included.

  The protein 'sample is not particularly limited as long as it contains a protein. Specifically, cells, tissues, blood and the like collected from a living body can be frozen and thawed, French press, glass beads, homogenizer, Unfractionated sample obtained by crushing treatment using a sonication device, etc., and cell membrane fraction sample obtained by subjecting such unfractionated sample to fractionation by fractional centrifugation, sucrose density gradient centrifugation, etc. And cytoplasmic fraction samples. The protein 'sample may be the same sample as the protein sample in step (a), but a different sample is preferable because information on more peptide peaks can be stored in the database.

  After the protein 'sample is fragmented with a protein digestion enzyme and the peptide' group is prepared, analysis is performed using LC-MS / MS, and the elution time of the peptide peak 'group and the standard elution of the peptide peak' group are analyzed. The detailed description until the acquisition of the MRM transition corresponding to the time and the peak intensity is as described above in the step (a). The conditions such as the fragmentation treatment with the protein digestive enzyme in the step (b), the analysis using LC-MS / MS, the identification and selection of the peptide peak are preferably the same as the conditions in the step (a).

Step (c)
In step (c), a peptide peak common to the peptide peak 'group is selected from the peptide peak group as a peptide peak group for standardization. The peptide peak group for standardization is selected using relational database software such as Access software (manufactured by Microsoft), MRM transition (mass-to-charge ratio [m / z] of Q1 and Q3) in the peptide peak group and the above. This can be done by comparing the MRM transitions in the 'peptide peak' group and selecting peptide peaks with the same (same) MRM transition.

Step (d)
In step (d), based on the standard elution time of the peptide peak group for standardization selected in step (c), the standard elution time of the peptide peak group and the elution time of the peptide peak ′ group are aligned, The elution time of the peptide peak 'group is normalized to the standard elution time of the peptide peak group, and the standard elution time of the peptide peak' group is obtained. As a method for normalizing the elution time of the peptide peak 'group to the standard elution time of the peptide peak group, the peptide is optimized so as to be optimized based on the standard elution time of the peptide peak group for standardization in the peptide peak group. Any method can be used as long as it can shift the elution time of the peak 'group (actually measured value) and standardize it to the standard elution time of the peptide peak group. For example, Origin 9 software (OriginLab), Excel (Microsoft), etc. Using software, for the peptide peak group for standardization among the peptide peak 'group, the shift time is calculated by smoothing using a percentile filter (100 points, 50%), and the elution time is shifted based on the calculated time. In addition, for peptide peaks other than the peptide peak group for standardization among the peptide peaks Depending on the shift amount of standardization for peptide peaks (e.g., calculates the shift time using a linear correction), it shifts the elution time based on the calculated shift time can be a method.

Step (e)
In step (e), the standard elution time of the peptide peak 'group obtained in step (d) is incorporated into the standard elution time of the peptide peak group and stored in the database as the standard elution time of the new peptide peak group. . That is, in step (e), the standard elution time of the peptide peak group is stored in the standard elution time of the peptide peak group (the elution time of the peptide peak group is normalized to the standard elution time of the peptide peak group). Create a database of accumulated standard elution times.

Step (f)
In step (f), the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak 'group are stored in the database. That is, in step (f), in addition to the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak group, the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak 'group were stored in the database and accumulated. Store MRM transition and peak intensity corresponding to standard elution time. Here, among the peak intensities corresponding to the standard elution time of the peptide peak 'group, the peak intensity of the standardizing peptide peak group is an average value or total value with the peak intensity of the standardizing peptide peak group stored in the database. In addition, the two values may be compared and the larger value may be selected and stored in the database, or both may be accumulated and stored in the database in order to calculate the total value as necessary.

  By repeating steps (b) to (f), a database with an increased accumulation level of accumulated standard elution time can be created, so steps (b) to (f) are repeated at least twice. Preferably, as at least 2 times, for example, 2 to 200 times, 2 to 100 times, 2 to 50 times, 2 to 25 times, 2 to 10 times, 10 to 200 times, 10 to 100 times, 10 to 50 times 10 to 25 times, 25 to 200 times, 25 to 100 times, 25 to 50 times, 50 to 200 times, 50 to 100 times, etc., but 2 to 25 times are more preferable. In addition, when repeating steps (b) to (f), the protein peak for obtaining the MRM transition and peak intensity corresponding to the elution time of the peptide peak 'group obtained in step (b) and the elution time, In order to efficiently increase the accumulation level of the accumulated standard elution time, different samples are preferable for each repetition.

  Analysis using LC-MS / MS for one or more target proteins in one or more samples using a database created using the method for creating this database (hereinafter sometimes referred to as “the present database”) When carried out according to a method comprising the following steps (A) to (E) (hereinafter sometimes referred to as “the present analysis method”), the peptide peak of the peptide constituting the target protein can be identified.

Step (A)
In the step (A), the target protein group in the sample is fragmented with a protein digestive enzyme to prepare the target peptide group.

  The sample is not particularly limited as long as it contains a target protein. Specifically, cells, tissues, blood, etc. collected from a living body can be frozen and thawed, French press, glass beads, homogenizer, ultrasonic wave. Unfractionated samples obtained by crushing treatment using a crushing device, etc., and cell membrane fraction samples obtained by fractionating such unfractionated samples by fractional centrifugation, sucrose density gradient centrifugation, etc. A cytoplasmic fraction sample can be mentioned.

  The protein digestion enzyme is not particularly limited as long as it is an enzyme that can be digested (cut) at a specific amino acid site of a protein to prepare a fragmented peptide. Specifically, trypsin, chymotrypsin, endoproteinase Glu-C, Mention may be made of endoproteinase Lys-C, endoproteinase Arg-C, endoproteinase Asn-C, lysyl endopeptidase, and clostripain. The fragmentation treatment with a protein digestion enzyme is not particularly limited as long as it is a fragmentation treatment with one or a plurality of protein digestion enzymes, but from the viewpoint of efficient fragmentation treatment, a fragmentation treatment with a plurality of the above protein digestion enzymes is preferable. Specifically, a fragmentation treatment using a combination of trypsin and lysyl endopeptidase can be preferably exemplified. As the fragmentation treatment conditions by the protein digestion enzyme, conditions such as temperature and time can be appropriately selected depending on the properties of the protein digestion enzyme to be used. It is preferable that it is 16 to 24 hours at 36 to 37 ° C. When lysyl endopeptidase is used, it is preferably 1 to 36 hours at 15 to 45 ° C, more preferably 2 to 4 hours at 15 to 25 ° C. When trypsin and lysyl endopeptidase are used in combination, it is preferably 1 to 36 hours at 15 to 45 ° C, more preferably 10 to 14 hours at 20 to 30 ° C.

Process (B)
In step (B), analysis using LC-MS / MS is performed using the target peptide group prepared in step (A), and the elution time and mass-to-charge ratio (m / z) of the peptide peak in liquid chromatography ) (MRM transition corresponding to peptide peak elution time) and data including MRM chromatogram information. Here, the “data including MRM chromatogram information” means data that can extract the MRM chromatogram of the peptide peak, and specifically, the peptide peak in the MRM transition corresponding to the elution time of the peptide peak. Data that can extract a (two-dimensional) chromatogram composed of elution time and peak intensity.

  In the analysis using LC-MS / MS, peptide groups are first separated by LC (liquid chromatography) of LC-MS / MS. Here, as liquid chromatography, specifically, cation exchange chromatography that performs separation using the difference in the charge of the peptide or reverse phase chromatography that performs separation using the difference in the hydrophobicity of the peptide. It may be mentioned, and may be a combination of both.

  Next, tandem mass spectrometry (MS / MS) is performed on each separated peptide. The ionization method in mass spectrometry is preferably an electrospray ionization method (ESI method), which is a soft ionization method. In mass spectrometry, peptides ionized by various ionization methods are separated according to mass by an analyzer. Examples of the analyzer include a magnetic mass separator (Sector MS), a quadrupole mass separator (QMS), a time-of-flight mass separator (TOFMS), and a Fourier transform ion cyclotron mass separator (FT-ICRMS). Or a combination thereof.

  As a method from detection of ionized peptides to acquisition of MS / MS data, a method in an automated measurement mode is preferable. Specifically, an IDA (Information Dependent Acquisition) measurement method, a DDA (Data Dependent Acquisition) measurement method, etc. Can be mentioned. In addition, data including MRM chromatogram information can be acquired using an MRM measurement method or a SWATH-MS measurement method. The obtained MS / MS data is imported into protein identification software such as Protein Pilot, Peak View, MASCOT, etc., and peptide groups are identified using protein databases such as UniProt, EMBL, Swiss-Prot. Some of the identified peptide groups have a peptide peak with low reliability, so those with a highly reliable peptide peak from among the peptide peaks of the identified peptide group (peptide peak group) As a highly reliable peptide peak, MS / MS data of the peptide group identified by the above software is converted into an ion library file (text file) and exported to Access (manufactured by Microsoft). Thus, it is possible to select based on a peptide confidence score (peptide confidence score) which is an index indicating peptide reliability (the reliability score when the reliability is 100% is shown as 1.00), for example, A peptide with a score greater than 0.85, a peptide greater than 0.90, a peptide greater than 0.92. Peptides greater than 0.94, peptides greater than 0.95, peptides greater than 0.96, peptides greater than 0.97, peptides greater than 0.98, peptides greater than 0.99, and the like. More than 99 peptides are more preferred.

Process (C)
In the step (C), the target peptide peak group is identified based on the mass-to-charge ratio (m / z) of the peptide peak acquired in the step (B), and stored in the database from the target peptide peak group. A peptide peak common to the peptide peak group is selected as the target peptide peak group for standardization. The target peptide peak group for standardization is selected by using software such as Access software (manufactured by Microsoft), and the MRM transition (the mass-to-charge ratio of Q1 and Q3 [m / Z]) and the MRM transition in the target peptide peak group, and a peptide peak having the same (same) MRM transition can be selected.

Process (D)
In step (D), based on the elution time of the target peptide peak group for standardization selected in step (C), the elution time of the target peptide peak group and the standard elution time of the peptide peak group stored in this database The standard elution time of the peptide peak group stored in the database is shifted to the elution time of the target peptide peak group, the shifted standard elution time, and the MRM transition corresponding to the shifted standard elution time and The peak intensity is acquired as a reconstructed ion library. As a method of shifting the standard elution time of the peptide peak group stored in the database to the elution time of the target peptide peak group, based on the elution time of the target peptide peak group for standardization in the target peptide peak group, Any method can be used as long as the standard elution time of the peptide peak group in this database is shifted so that it can be optimized, and the actual value can be converted to the elution time of the target peptide peak group. For example, Origin 9 software (manufactured by OriginLab), Excel Using software such as Microsoft (manufactured by Microsoft), the shift time is calculated by smoothing using the percentile filter (100 points, 50%) for the peptide peak group for standardization among the peptide peak groups in this database. The standard elution time is shifted based on the measured time and the database For peptide peaks other than the target peptide peak group for standardization in the peptide peak group, the calculated shift time according to the shift amount of the target peptide peak for standardization nearby (for example, the shift time is calculated using linear correction). A method of shifting the elution time based on the above can be mentioned. The shifted standard elution time is imported into Access (manufactured by Microsoft) and further reconstituted with the MRM transition and peak intensity corresponding to the shifted standard elution time. Data including MRM transitions and peak intensities corresponding to shifted standard elution times).

Process (E)
In step (E), the MRM chromatogram of the peptide peak is extracted from the data including the MRM chromatogram information acquired in step (B) using the reconstructed ion library acquired in step (D), and the MRM chromatogram Identify peptide groups of interest from the gram.

  In order to identify the target peptide peak group from the MRM chromatogram of the peptide peak, first, from the data including the MRM chromatogram information, the peptide peak elution time and the peak intensity in each MRM transition of the peptide peak derived from the target peptide To create a (two-dimensional) chromatogram. Next, from these chromatograms, peptide peaks having the same elution time as the standard elution time shifted in the reconstructed ion library (standard elution time converted so that the elution time of the target peptide peak group becomes the reference). And a peptide peak having an elution time in common with the shifted standard elution time is identified as the target peptide peak. Here, the “common elution time” includes the same elution time as the shifted standard elution time, as well as an elution time in a range including the shifted standard elution time, for example, the shifted standard elution time ± 2.0 minutes, Shifted standard elution time ± 1.5 min, shifted standard elution time ± 1.0 min, shifted standard elution time ± 0.5 min, shifted standard elution time ± 0.3 min, shifted standard elution time ± 0.2 minutes, shifted standard elution time ± 0.1 minutes, etc. are also included. If this database stores relevant information such as the UniProt accession number corresponding to the standard elution time of peptide peaks and peptide amino acid sequences and valences that contain information related to peptide modification, the relevant information will be used as the basis. Furthermore, the amino acid sequence of the peptide corresponding to the target peptide peak, that is, the peptide constituting the target protein can be specified.

  In this analysis method, in order to quantify the peptide peak of the peptide constituting the target protein among a plurality of samples, the ratio of the peak area to the peak intensity is further selected from the target peptide peak group identified in step (E). Based on the above, the step (F) of selecting the peptide peak for quantification of the target peptide peak group, and the peptide peak area for quantification of the target peptide peak group selected in the step (F) were calculated for a plurality of samples and calculated. What provided the process (G) which quantifies the peptide peak of the peptide which comprises the target protein between several samples from ratio of a peak area is preferable.

Process (F)
In the step (F), a peptide peak for quantification of the target peptide peak group is selected. The peptide peak for quantification of the target peptide peak group can be selected from the target peptide peak group identified in the step (E) using the ratio of the peak area and the peak intensity as an index, specifically, For the target peptide peak identified in step (E), “(d) the peak area obtained from the chromatogram of the MRM transition that gives the maximum peak intensity among the MRM transitions of the target peptide peak stored in the database”; “(C) the total peptide peak area obtained from the MRM transition chromatogram of the target peptide peak stored in the database”, “(a) the maximum peak intensity in the MRM transition that gives the maximum peak intensity”, “(B) In the MRM transition used for the sum of the peak areas The peak area obtained from the chromatogram of the MRM transition that gives the maximum peak intensity among the MRM transitions of the target peptide peak stored in the database (c) / (c) Total of peptide peak areas obtained from chromatogram of MRM transition of target peptide peak stored in database] / [(a) Maximum peak intensity in MRM transition that gives the maximum peak intensity / (b) Total of peak area The value (index value) is calculated by inputting the value into the “total sum of peak intensities in MRM transition used in the above”, and the calculated index value is in the range of 0.25 to 4.0, preferably 0.33 to 3.0. A target peptide peak showing a range, more preferably a range of 0.5 to 2.0. Selected as an amount for peptide peaks. The MRM transitions of the target peptide peak stored in the database used for the total peptide peak area (c) and the peak intensity total (b) may be MRM transitions of all target peptide peaks. An MRM transition that gives a peak intensity exceeding a certain ratio with respect to the peak intensity may be selected. In this case, “exceeding a certain ratio with respect to the maximum peak intensity” is, for example, 5% with respect to the maximum peak intensity. Exceeding 10%, exceeding 15%, exceeding 20%, exceeding 25%, exceeding 30%, etc., but preferably exceeding 20% with respect to the maximum peak intensity. Further, the peptide peak for quantification is preferably selected based on the number of MRM transitions. For example, a peptide peak having an MRM transition number of 2 or more, 3 or more, 4 or more, 5 or more is selected as the peptide peak for quantification. However, it is more preferable to select a peptide peak having an MRM transition number of 3 or more as a peptide peak for quantification.

Process (G)
In the step (G), as a method for quantifying the peptide peak of the peptide constituting the target protein among a plurality of samples from the ratio of the calculated peak areas, the peak of the peptide peak of the peptide constituting the target protein in a specific sample is used. ¹⁵ N, ¹³ C, ¹⁸ O, and ² H in addition to a method for relatively quantifying the change in area and the peak area of the peptide peak of the peptide constituting the target protein in a sample different from the specific sample A peptide-labeled stable isotope-labeled target peptide that constitutes a target protein with any one or more stable isotope-labeled elements is prepared, and this stable isotope-labeled target peptide is used as an internal standard for a specific sample. The peak area of the peptide peak of the peptide constituting the target protein, and the specific sample Includes a method for absolute quantification of a change in the peak area of the peptide peak of the peptide constituting the target protein in different samples.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Result 1: Overview of Method for Creating MRM Array Database (Database of the Present Invention)]
An outline of the database creation method of the present invention is shown in FIGS. First, a sample for mass spectrometry is prepared from the first protein sample (“Sample # 1” in FIG. 1A) (for details, refer to [Method 1: Preparation of sample to be analyzed by LC-MS / MS] described later). Mass spectrometry using LC-MS / MS is performed, and MS / MS data of the first sample peptide peak (group) constituting the first protein sample is obtained by IDA (Information Dependent Acquisition) measurement (details will be described later) [See Method 2: LC-MS / MS]. Subsequently, the first sample peptide group is identified using Protein Pilot, Peak View software, or the like based on the MS / MS data, and the MS / MS data is converted into an ion library file. Next, a highly reliable peptide peak group (for example, in this example, a confidence score> 0.99 was used as a selection criterion) and a more reliable average elution time (in this example, Peptide peak group having standard deviation [SD] <0.2 min of elution time of the same peptide as a selection criterion) is selected, and the elution time of such peptide peak group is defined as standard elution time (standard RT, nRT) Save (store) in database. Next, the second protein sample ("Sample # 2" in FIG. 1A) is also analyzed in the same manner as the first protein sample, the second sample peptide peak group is identified and selected, and the measured RT2 of the second sample peptide peak group is identified. To get. Thereafter, a peptide peak common to the second sample peptide peak group is selected as a standardization peptide peak group from the first sample peptide peak group stored in the database. Based on the nRT of the peptide peak group for standardization, the measured RT2 of the peptide peak for standardization in the second sample peptide peak group is shifted so as to optimize the alignment (for example, a percentile filter is used in this embodiment). Calculate the shift time by smoothing). Peptide peaks other than the standardization peptide peak in the second sample peptide peak group are shifted according to the shift amount of the nearby standardization peptide peak (for example, in this embodiment, the shift time is calculated using linear correction). With the above shift, the measured RT2 is standardized (converted) to nRT with the measured RT2 as nRT2 for all second sample peptide peak groups. By storing nRT2 of the second sample peptide peak group in a database, a database of accumulated nRT (nRT + nRT2) is created. Simultaneously with the accumulation of nRT, the MRM transition, peak intensity and related information (valence, UniProt accession number, peptide amino acid sequence including information on peptide modification) are also stored (FIG. 1B). Further, the third protein sample (“Sample # 3” in FIG. 1A) is also analyzed in the same manner as the first protein sample and the second protein sample, and the third sample peptide peak group is identified and selected. The measured RT3 of the 3-sample peptide peak group is acquired, normalized to nRT3, and stored in a database, thereby increasing the accumulation level of accumulated nRT (nRT + nRT2 + nRT3). From the fourth protein sample onward, a highly reliable average nRT is selected from the database at the time of alignment for each peptide peak, and compared with the measured RT (for example, in this example, the standard deviation of RT [SD] <0.2 minutes as selection criteria).

[Result 2: Database creation method of the present invention]
The results of creating the database of the present invention using human liver microsomes (HLM) (XTreme 200, manufactured by XenoTech) are shown in FIGS. RT of three fractions obtained by fractionating tryptic peptides of HLM on a reverse phase column under alkaline conditions ("red (fraction 1), blue (fraction 2), and green (fraction 3) in FIG. 1C") )) And RT of the unfractionated HLM as a control, the RT of the peptide peaks identified in the three fractions despite continuous measurement was different from the unfractionated HLM. The time difference was different for each fraction (FIG. 1C). The nRT conversion process is shown for RT of one kind of fraction (red [fraction 1]). The nRT of 6858 common peptide peaks (standardization peptide peaks) and the measured RT of standardization peptide peaks of fraction 1 in the database of the present invention (a database in which data of 23 types of samples described later are accumulated) constructed in advance. The time difference was plotted (FIG. 1D, red dot), and the amount of shift time was calculated by smoothing (FIG. 1D, green line). The difference between the nRT converted from the measured RT of the standardization peptide peak in fraction 1 and the nRT of the standardization peptide peak in the database is within 0.2 min for the 98.3% standardization peptide peak. (FIG. 1E) This result shows that the error can be converted to nRT within 0.2 minutes. Similar results were obtained when the LC solvent was replaced anew or when the intracellular fraction sample of cultured cells was analyzed.

  Further, 23 types of samples (5 unfractionated HLM data sets [XTreme 200, manufactured by XenoTech), 10 HLM fraction data sets [XTreme 200, manufactured by XenoTech], cell lines [HEK293, Caco2 and BeWo] ] And 8 synthetic data sets [Ohtsuki, S., et al. J Pharm Sci 100, 3547-3559 (2011), Shawahna, R., et al Mol Pharm 8, 1332-1341 (2011), Uchida, Y., et al. J Neurochem 117, 333-345 (2011)]. (See [Method 3: Accumulation of nRT data in the database of the present invention] described later for details). As a result, a database including nRT and MRM transition information of 107,715 peptide peaks could be created. Such peptide peaks are characterized by a UniProt accession number and a peptide sequence and valency containing information regarding the modification of the precursor peptide. In addition, there were 54,980 peptide peaks in which data from two or more experiments were accumulated, and 97.0% of them showed that the standard deviation (SD) was within 0.2 minutes. Select peptide peaks for which RT was SD <0.2 min and peptide peaks for which RT was detected in one experiment (total 106,074 peptide peaks), and the nRT, MRM transition and peak intensity of these peptide peaks. In addition, a database containing related information (valence, UniProt accession number, peptide amino acid sequence including information on peptide modification) was used for subsequent analysis.

[Result 3: Outline of ion library reconstruction method using database of the present invention]
An outline of a method for reconstructing an ion library using the database of the present invention is shown in FIG. First, IDA analysis using LC-MS / MS is performed using a measurement sample containing the target protein to obtain an ion library file. Next, a target peptide peak group with high reliability (for example, in this example, a confidence score> 0.99 is selected as a selection criterion) is selected and stored in the target peptide peak group and the database of the present invention. Peptide peaks that are common peptide peaks and have high reliability of RT values based on both RTs (for example, in this example, standard deviation [SD] <0.2 Minutes as a selection criterion) is selected as the target peptide peak group for standardization. Based on the RT of the target peptide peak group for standardization, the nRT of the standard peptide peak group stored in the database is shifted and corrected to convert the nRT to RT based on the actual measurement value and create a reconstructed ion library And used for SWATH quantitative analysis.

[Result 4: Reconstruction of ion library using database of the present invention]
An IDA analysis using LC-MS / MS is performed using HLM, and after obtaining an ion library file, a reconstructed ion library is created using the database of the present invention (for details, see [Method 4: Reconstruction of ion library file based on database]), the difference between the measured RT and the shifted nRT was measured (FIG. 2B). Compare the measured value RT with the shifted nRT for the peak of the target peptide group (1585 peptides) having a confidence score of 0.90 to 0.99 that was not used as the elution time for reconstruction. The width was verified. As a result, it was shown that there were 95.4% (1512) target peptide peaks having a difference width between them of 0.2 minutes or less (FIG. 2B). For the target peptide peak group (12863 peptides) having a confidence score> 0.99 used as the elution time for reconstruction, RT of 12784 peptide peaks (99.4%) was 0.2 minutes. It was shown that the difference was within. These results indicate that the actual measured value RT corresponding to the shifted nRT can be sufficiently specified, and therefore the target peptide peak on the chromatogram can be identified using the shifted nRT.

[Result 5: SWATH quantitative analysis 1 using the database of the present invention]
Next, it was examined whether the peak area of the peptide peak identified using the database of the present invention could be quantified reproducibly. A part of the measurement target sample was subjected to IDA analysis, the target peptide was identified, and the ion library was reconstructed using the elution time of the common peptide peak (standardization peptide peak) with the target peptide using the database of the present invention. Further, the sample to be measured was subjected to SWATH analysis, and the data acquired by SWATH was analyzed using an ion library obtained by reconstructing the sample, and the peak area of the target peptide peak was quantified. After 1.5 months, similar analysis was performed using similar samples, and the peak areas of the two were compared. The peak area was standardized by the total peak area of all target peptide peak groups. As a result, as shown in FIG. 2D, it was shown that the target peptide peak that can be quantified with good reproducibility within the difference width within 2 times between the two measurement target samples remains at 61.1% (59553/97512). In particular, many of the target peptide peaks exhibiting a low peak area were quantified with a difference exceeding twice, indicating that there was a problem in reproducibility. As a cause of reducing the reproducibility, it was considered that among the target peptide peaks identified using the database of the present invention, the target peptide peak below the detection limit was also quantified. For this reason, it was thought that it was necessary to validate the target peptide peak which can be quantified.

The present inventors have developed a simple index for peak validation (see [Method 5: Peptide peak validation] described below for details). That is, the peak area obtained from the chromatogram of the MRM transition that gives the maximum peak intensity among the MRM transitions of the target peptide peak stored in the present database / (c) with respect to the database of the present invention Total sum of peptide peak areas obtained from chromatograms of MRM transitions of target peptide peaks stored in this database] / [(a) Maximum peak intensity in MRM transition giving maximum peak intensity / (b) of peak area The sum of peak intensities in MRM transitions used for summation was calculated for each MRM transition, and target peptide peaks in the range of 0.5 to 2 were extracted as effective peaks. In this example, the MRM transition of the target peptide peak stored in the database used for the total peptide peak area (c) and peak intensity total (b) is 20% of the maximum peak intensity. An MRM transition that gave a peak intensity that exceeded was selected (see [Method 4: Reconstruction of ion library file based on database of the present invention] described later). When a peak is extracted using the validation index, the ratio of peptide peaks whose peak area difference width is within 2 times increases from 61.1% to 87.4% (37707/43129), and the peak area difference width is The ratio of peptide peaks within 1.5-fold was shown to increase from 47.5% to 74.1% (FIG. 2E). This result shows that the validation of the peptide peak is effective. Furthermore, in order to increase the proportion of target peptides that can be quantified with good quantitativeness, when peptide peaks with an MRM transition number of 3 or more are extracted, the proportion of peptides with a peak area difference difference of 2 times or less is 87.4% to 90.3%. It was shown to increase to (32235/35692) (FIG. 2F). Even after such peptide peak validation, the number of peptides that can be quantified is 4.23 times (32235/7615: comparison between FIG. 2C and F) more than when using the standard SWATH quantification method. It was. In addition, when focusing on the lower limit of the peak area of the target peptide that can be quantified, it was shown that even a level that is 1/10 lower than when using the standard SWATH quantification method can be quantified (10 ³ count in FIG. 2F and the figure). 10 ⁴ count comparison of 2C). These results indicate that peptide peaks that can be quantified can be efficiently extracted by identifying the peptide peaks to be measured using the database of the present invention and performing validation of the peptide peaks from the identified peptide peaks. .

[Result 6: SWATH quantitative analysis 2 using the database of the present invention]
Next, a model sample spiked with a synthetic peptide (for details, see [Method 6: Peptide peak using the database of the present invention described later) is used to determine whether the increase or decrease in the area of the peptide peak extracted by validation can be sufficiently detected. Quantitative verification]]). A sample spiked with 0.1 fmol of synthetic peptide and a sample spiked with 25 fmol of synthetic peptide were examined for the presence or absence of an increase. When standard SWATH quantification was used, a synthetic peptide peak that could detect an increase was detected. The number was 171 (54.8% of the total number of registered peaks [312]), but quantified using an ion library reconstructed from the database of the present invention and extracted by validation (hereinafter referred to as “the present invention”). The number of synthetic peptide peaks for which an increase could be detected increased to 263 (84.3% of the total number of registered peaks [312]) (FIG. 3A). . Similar results were obtained even when analysis was performed using other combinations with different synthetic peptide ratios. For example, a sample spiked with 0.5 fmol of synthetic peptide and 25 fmol of synthetic peptide were spiked. In comparison with the sample, when standard SWATH quantification was used, the number of synthetic peptide peaks that could detect an increase was 171 (54.8% of the total number of registered peaks [312]), whereas the present invention The number of synthetic peptide peaks for which an increase was detected was increased to 264 (84.6% of the total number of registered peaks [312]), and 5 fmol of synthetic peptide was detected. Synthetic peptides that were able to detect an increase between the spiked sample and the sample spiked with 25 fmol of synthetic peptide using standard SWATH quantification. The number of peak peaks was 166 (53.2% of the total number of registered peaks [312]), whereas when the SWATH quantification using the database of the present invention was used, the number of synthetic peptide peaks that could detect an increase was It increased to 238 (76.2% of the total number of registered peaks [312]) (FIG. 3A). These results indicate that the synthetic peptide is identified using the database of the present invention, and the peptide peak extracted by validation of the identified synthetic peptide peak can detect the increase or decrease in the peak area with high sensitivity.

  Next, it was examined whether the peptide peak extracted by the above-described validation can accurately quantify the increase or decrease in the peak area. The area ratio of peptide peaks between a sample spiked with 0.5 fmol of synthetic peptide and a sample spiked with 5 fmol of synthetic peptide (for details, see [Method 6: Quantification of peptide peak using database of the present invention described later) Comparison])) showed a 10.4 fold difference (FIG. 3C). That is, detection was possible with a peak area ratio corresponding to the ratio of the amount of the synthetic peptide. This result indicates that the peptide of the present invention is identified by using the database of the present invention, and the peptide peak extracted by validation of the identified peptide peak can be detected with sufficient accuracy. Even when the standard SWATH quantification method is used, the peptide peak area ratio is 9.9 times different between the sample spiked with 0.5 fmol of the synthetic peptide and the sample spiked with 5 fmol of the synthetic peptide. However, the SWATH quantification method using the database of the present invention is excellent in that it can accurately detect the increase and decrease of peptide peaks that could not be identified using the standard SWATH quantification method.

[Method 1: Preparation of sample to be analyzed by LC-MS / MS]
Samples to be analyzed by LC-MS / MS are literature (Ohtsuki, S., et al. Drug Metab Dispos 40, 83-92 (2012), Ohtsuki, S., et al. J Pharm Sci 100, 3547-3559 ( 2011), Yoneyama, T., et al. J Proteome Res 12, 753-762 (2013)). The preparation method is shown below. First, a protein sample (50 μg) was suspended in a suspension containing 7 M guanidine hydrochloride and 10 mM EDTA. The sample was reduced with DTT (dithiothreitol) for 60 minutes at room temperature in the presence of nitrogen, and then S-carbamoylmethylated with iodoacetamide for 60 minutes at room temperature. The alkylated protein was precipitated with a mixture of methanol and chloroform. This precipitate was dissolved in 6M urea and diluted with 100 mM Tris-HCl (pH 8.0). After diluting the sample to 1 M urea, trypsin digested with lysyl endopeptidase at the enzyme / substrate ratio of 1: 100 at 25 ° C. for 3 hours and then treated with tosylphenylalanyl chloromethyl ketone is then enzyme / substrate Was digested at 37 ° C. for 16 hours. The sample was desalted using a GL-SDB chip and a GL-GC chip (manufactured by GL Sciences Inc.), evaporated, dissolved in 0.1% formic acid, and a sample was prepared.

[Method 2: LC-MS / MS]
A nanoLC system (Ultimate 3000 RSLCnano; manufactured by DIONEX) was connected to a nano-electrospray ionization mass spectrometer (TripleTOF 5600; manufactured by ABSCIEX) operated in the positive ionization mode to analyze a sample containing the target protein. . The parameter values of ion source gas, curtain gas, ion spray voltage, interface heater temperature, and declustering potential were 20, 20, 2300, 150, and 80, respectively. Rolling collision energy is used at an accumulation time of 50 microseconds, collision energy dispersion coefficient 5, ion emission delay 30 and ion emission width 15, and precursor ions (Q1) are 300 to 1008 and product ions (Q3) are 100 to 1600. Scan and IDA method was performed. The maximum number of candidate precursor ions for observing product ions was 20 ions / cycle. Analyzed ions were excluded for 10 seconds. The accumulation time was set to 50 microseconds, the SWATH window of precursor ions was set to a mass-to-charge ratio of 300 to 1008 13 Da (including 1 Da overlap), and the SWATH-MS acquisition method was performed. Product ions were scanned from 100 to 1600 using the rolling collision energy with the same settings as the IDA measurement. The cycle time was 3.05 seconds. Using a C18 column (Acclaim PepMap RSLC C18, 2 μm, 100 mm, inner diameter 75 μm × 25 cm, manufactured by DIONEX) and a trap column (inner diameter 100 μm × 2 cm, packed with Acclaim PepMap100 C18, 5 μm, 100 mm, manufactured by DIONEX) at 40 ° C. NanoLC was performed. Apply a linear gradient containing 1-25% and 25-50% acetonitrile in 0.1% formic acid at a flow rate of 300 nL / min for 60 minutes and 15 minutes (Figures 1 and 2) or 40 minutes and 10 minutes. (FIG. 3), the target peptide was eluted.

[Method 3: Accumulation of nRT data in the database of the present invention]
MS / MS data obtained by IDA measurement using the above 23 types of samples and 2 types of synthetic peptide data sets are imported into Protein Pilot (ABSCIEX), and UniProt human protein database is searched to find the target peptide. Identified. The result file (corresponding to [group file] in FIG. 1) was imported into Peak View using SWATH MicroApp (ABSCIEX), and the ion library file (text file) was exported to Access (Microsoft). Extracts transition data with a peptide confidence score exceeding 0.99, links each transition data with a unique transition name and a unique peptide peak name, and stores it in Microsoft SQL Server Express as a transition database did. In order to reduce the database size, the transition data was integrated with a unique transition name by summing the relative intensities. The RT average and standard deviation (SD) of each peptide peak was calculated in Access, and the RT average with SD <0.2 min was used for RT alignment. List all RTs and nRTs in Access, export all RTs sorted by RTs and RT-nRTs of standardized peptide peaks to Origin 9 software (OriginLab) for correction (smoothing and interpolation) Went. Smoothing was performed using a percentile filter (100 points, 50%), and linear interpolation was performed using smoothed data. The interpolated data was stored in the nRT database in Access. NRT obtained from various samples was stored in the nRT database. In addition, in order to compare with RT of IDA data, the average of nRT and SD of each peptide peak are calculated by Access, the average of RT with SD <0.2 minutes, or the RT of peptide detected in one experiment. (RT for a total of 106,074 peptides).

[Method 4: Reconstruction of ion library file based on database of the present invention]
Using HLM, analysis was performed by the IDA method in the course of the continuous SWATH acquisition method. An ion library file was created from the data file obtained by the IDA method and imported into Access. The average and SD of RT of the ion library and nRT of the nRT database were calculated, and all RTs and nRTs of overlapping peptide peaks with SD within 0.2 minutes were listed in Access. A list of all nRTs sorted based on nRT and RT-nRT of standardization peptide peaks was exported to Origin 9 software for smoothing and interpolation. Smoothing was performed using a percentile filter (100 points, 50%), and linear interpolation was performed using smoothed data. The shifted nRT was imported into Access and a reconstructed ion library file was created by integrating with the data in the transition database. Transitions with a maximum intensity greater than 20% at each peptide peak were selected for the reconstructed ion library file. The reconstructed ion library file was imported into Peak View equipped with SWATH MicroApp, and SWATH quantification was performed using the SWATH acquisition data. In order to obtain peak areas for all peptide peaks, the number of peptides, exclusion of modifications, exclusion of shared sequence selection, XIC extraction window, and XIC width were set to “999”, “off”, “off”, “1. “0 min” and “0.040 Da”. Since the difference between the measured RT and the shifted nRT was larger in FIG. 3 than in FIG. 2 due to the difference in gradient conditions, the XIC extraction window was set to 1.5 minutes in FIG. For standard SWATH quantification, 5 transitions were used to calculate the peak area. In quantification using the database of the present invention, the number of transitions was set to “999” in order to use all selected transitions in the reconstructed ion library.

[Method 5: Validation of peptide peak]
For peak validation, “relative peak intensity” in the transition database and “peak area” of each transition (ion) in the result file exported from Peak View software were used. The following procedure was performed using Access. For each peptide peak, the maximum peak intensity (a) in the MRM transition database, the sum of peak intensities (b), and the transition giving the maximum peak intensity are listed for each peptide peak. Next, in the result file of the sample exported from Peak View, the total peak area (c) and the peak area (d) of the transition that gives the maximum intensity in the transition database are listed for each peptide peak. For each peptide peak, the formula [(d) out of MRM transitions of the target peptide peak stored in the database, peak area obtained from the chromatogram of the MRM transition that gives the maximum peak intensity / (c) stored in the database. Sum of peptide peak areas obtained from chromatogram of MRM transition of target peptide peak to be obtained] / [(a) Maximum peak intensity in MRM transition that gives the maximum peak intensity / (b) MRM used for the sum of peak areas The index of validation was calculated by [total peak intensity in transition]. A peptide peak having an index value in the range of 0.5 to 2 was used for comparison.

[Method 6: Verification of peptide peak quantification using database of the present invention]
296 synthetic peptides (Ohtsuki, S., et al. J Pharm Sci 100, 3547-3559 (2011), Shawahna, R., et al. Mol Pharm 8, 1332- 1341 (2011), Uchida, Y., et al. J Neurochem 117, 333-345 (2011)) were added in 4 different amounts (0.1, 0.5, 5 and 25 fmol / injection). A model sample was prepared. With these peptides, 312 peptide peaks were detected in the model sample from 240 peptides in the database of the present invention. The sample was repeatedly analyzed by the SWATH-MS acquisition method (4 times). A model sample spiked with 25 fmol of the synthetic peptide was analyzed by the IDA method before the first sample and between the second and third repeats. The transition information of the synthetic peptide was deleted from the ion library file exported from Peak View, and a reconstructed peak file was created without using the synthetic peptide information. Due to the different slope conditions, the difference in RT was greater than in FIG. 2B and the time window of the detected peak was set to 1.5 minutes instead of 1.0 minute. The peak area data was imported into Marker View software (manufactured by ABSCIEX) and subjected to statistical analysis, and peptide peaks that significantly increased (p <0.01 times and> 1.5 times) were extracted. Peak validation was performed on these markedly increased peptide peaks. Since a part of the synthetic peptide can be used for measurement of the protein in the HLM, the peak area is the total area of the endogenous peptide and the added peptide. In order to exclude the amount of endogenous peptide, a peak area ratio of 0.5 or 5 fmol was calculated as follows.
Peak area ratio = (peak area of 0.5 or 5 fmol synthetic peptide−peak area of 0.1 fmol synthetic peptide) / (peak area of 25 fmol synthetic peptide−peak area of 0.1 fmol synthetic peptide)

  Since the present invention is a technique effective for biomarker search, it can be used in the medical, pharmaceutical and bio fields. In addition, SWATH data acquisition can only be done with ABSCIEX (the world's top share in mass spectrometry instrument) machine so far, so it is useful to put the algorithm of the database creation method of the present invention into the analysis software of ABSCIEX. It is. It is also expected that biosoftware companies will contribute to the development of analysis software by licensing the algorithm of the database creation method of the present invention.

Claims (5)

Peptide peak elution time for identifying and / or quantifying peptide peaks of peptides constituting a plurality of target proteins by analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS), and the peptide A method of creating a database storing MRM transitions corresponding to peak elution times and peak intensities, comprising the following steps (a) to (f).
(A) Corresponds to the standard elution time of the peptide peak group derived from the peptide group containing the peptide constituting the target protein, obtained by mass spectrometry using LC-MS / MS, and the standard elution time of the peptide peak group Obtaining MRM transitions and peak intensities and storing them in a database;
(B) The elution time of the peptide peak group derived from the peptide group including the peptide constituting the protein of interest obtained by mass spectrometry using LC-MS / MS, and the elution time of the peptide peak group Obtaining a MRM transition and a peak intensity corresponding to the above; (c) selecting a peptide peak common to the peptide peak 'group from the peptide peak group as a peptide peak group for standardization;
(D) Based on the standard elution time of the peptide peak group for standardization selected in the step (c), the standard elution time of the peptide peak group and the elution time of the peptide peak 'group are aligned, and the peptide peak' Normalizing the elution time of the group to the standard elution time of the peptide peak group, and obtaining the standard elution time of the peptide peak 'group;
(E) The step of incorporating the standard elution time of the peptide peak 'group obtained in step (d) into the standard elution time of the peptide peak group and storing it in the database as the standard elution time of a new peptide peak group;
(F) storing the MRM transition and peak intensity corresponding to the standard elution time of the peptide peak 'group in the database; The method according to claim 1, wherein the steps (b) to (f) are repeated 2 to 25 times. A method for identifying peptide peaks of peptides constituting one or more target proteins in one or more samples by analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS), comprising: A method comprising steps (A) to (E).
(A) A step of preparing a target peptide group by subjecting the target protein group in the sample to a fragmentation treatment with a protein digestive enzyme;
(B) Using the target peptide group prepared in step (A), analysis using a liquid chromatograph-tandem mass spectrometer (LC-MS / MS) is performed, and the peptide peak elution time, mass-to-charge ratio (m / Z), and obtaining data including MRM chromatogram information;
(C) The target peptide peak group is identified based on the mass-to-charge ratio (m / z) of the peptide peak obtained in step (B), and the method according to claim 1 or 2 is performed from the target peptide peak group. A step of selecting a peptide peak common to the peptide peak group stored in the database created by using the peptide peak group for standardization;
(D) Based on the elution time of the target peptide peak group for standardization selected in step (C), the elution time of the target peptide peak group and the database created using the method according to claim 1 or 2 are stored. The standard elution time of the peptide peak group stored in the database created using the method according to claim 1 is used as the elution time of the target peptide peak group. Acquiring a shifted standard elution time and an MRM transition and peak intensity corresponding to the shifted standard elution time as a reconstructed ion library;
(E) Using the reconstructed ion library acquired in step (D), the MRM chromatogram of the peptide peak is extracted from the data including the MRM chromatogram information acquired in step (B), and the target peptide is extracted from the MRM chromatogram. Identifying a peak group; Furthermore, the method of Claim 3 provided with the following process (F) and (G) for quantifying the peptide peak of the peptide which comprises the target protein between several samples.
(F) From the target peptide peak group identified in step (E),
The peak area obtained from the chromatogram of the MRM transition that gives the maximum peak intensity among the MRM transitions of the target peptide peak stored in the database created by using the method according to claim 1 or 2, and The ratio of the peak area to the total peptide peak area obtained from the MRM transition chromatogram of the target peptide peak stored in the database created using the method described in 2.
For quantification of the target peptide peak group based on the ratio of the peak intensity ratio of the maximum peak intensity in the MRM transition that gives the maximum peak intensity to the total peak intensity in the MRM transition used for the sum of the peak areas. Selecting a peptide peak;
(G) The peptide peak area for quantification of the target peptide peak group selected in the step (F) is calculated for a plurality of samples, and the peptide peak of the peptide constituting the target protein among the plurality of samples is calculated from the ratio of the calculated peak areas. Quantifying The method according to claim 3 or 4 , wherein the fragmentation treatment by the protein digestion enzyme is a fragmentation treatment by combined use of trypsin and lysyl endopeptidase.