Application of database searching software in targeted proteomics analysis and analysis methods

Abstract

The present application relates to the technical field of proteomics analysis, and more particularly to the application of search software in targeted proteomics analysis and an analysis method. The present application proposes a targeted proteomics data processing method based on search software. By using the search software to process the mass spectrum data of PRM, the method realizes automatic operation by only importing the mass spectrum data of PRM and setting search parameters, and finally obtains accurate and reliable qualitative and quantitative results. The method does not require manual intervention during operation, reduces human error, makes up for the problems existing in the processing of targeted proteomics data by Skyline, and the results obtained by automatic processing of the search software are also superior to Skyline analysis in terms of accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of proteomics analysis, in particular to the application of search software in targeted proteomics analysis and an analysis method. BACKGROUND

[0002] Targeted proteomics is a method for detecting target proteins / polypeptides, which can realize high specificity and high accuracy of target protein quantification by selecting and detecting signals related to target proteins / polypeptides and ignoring other irrelevant signals. At present, targeted proteomics has been widely applied in the research of systems biology and biomedical science. Targeted proteomics technology mainly includes two methods of SRM / MRM (Selected / Multiple reaction monitoring) and PRM (Parallel reaction monitoring). PRM technology is proposed on the basis of SRM / MRM, which combines the high selectivity of quadrupole and the high resolution and high accuracy characteristics of Orbitrap, can independently identify the secondary spectrum, and the method process is more convenient. Compared with traditional SRM / MRM, PRM has better anti-interference ability and detection sensitivity in complex background, and can simultaneously detect hundreds of proteins. Therefore, PRM is a new type of targeted proteomics detection method with more advantages and potential, which is called the upgraded version of SRM / MRM, and is a widely used targeted proteomics technology at present.

[0003] At present, the software widely used for data processing of targeted proteomics, especially PRM, is Skyline. Before Skyline processes PRM data, a reference database needs to be imported first, and then the target protein / polypeptide is spectrum matched based on the database, and quantified based on the polypeptide fragment ion peak area. The algorithm of Skyline spectrum matching is relatively simple, mainly focusing on the fragment ions with high abundance. For complex samples, the data after automatic processing still needs to be verified manually in the later stage, and the fragment ions with interference are removed. Only the fragment ions with symmetrical peak type will be selected for quantification. In addition, the boundary judgment error of Skyline software automatic integration also needs to be adjusted manually to ensure that the boundaries of the same target polypeptide integration in the same batch of samples are consistent. These manual inspection and adjustment processes are time-consuming and laborious, especially when a large number of samples are processed, which is a heavy work. In addition, the inspection based on human judgment also inevitably exists human error. These problems have become one of the key problems that need to be solved in the current targeted proteomics based on PRM.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The application aims at replacing Skyline in processing PRM data by using search software, avoiding manual intervention, reducing human error, making up for the problems in processing targeted proteomics data by Skyline, improving the accuracy of analysis, and providing a specific method for processing PRM data by using search software.

[0006] In order to solve the above technical problems and achieve the above purposes, the application provides the following technical solutions.

[0007] In a first aspect, the application provides an application of search software in targeted proteomics analysis, wherein the search software is used for analyzing PRM mass spectrum, the mass spectrum is obtained by a mass spectrometry device containing a quadrupole rod and a high-resolution detector, and includes a primary full-scan mass spectrum and a secondary PRM mass spectrum.

[0008] The acquisition parameters of the primary full-scan mass spectrum include:

[0009] (a) positive ion mode, (b) Orbitrap resolution greater than 10000, (c) scanning range of 100-2000 m / z, (d) automatic gain control target (AGC target) greater than 8e3, (e) 10-30 ms of precursor ion accumulation time for scanning the primary mass spectrum.

[0010] The acquisition parameters of the secondary PRM mass spectrum include:

[0011] (a) positive ion mode, (b) Orbitrap resolution greater than 10000, (c) default charge state greater than 1, (d) 80-150 ms of ion accumulation time, (e) loop count greater than 20 times, (f) isolation window 1-3 m / z, (g) fragmentation energy (NCE) 25-40.

[0012] The primary full-scan mass spectrum and the secondary PRM mass spectrum are simultaneously acquired.

[0013] In an optional embodiment, the search software is selected from Proteomic Discoverer or MaxQuant.

[0014] In an optional embodiment, the acquisition parameters of the secondary PRM mass spectrum include: Orbitrap resolution of 15000, default charge state of 2, 100 ms of ion accumulation time, loop count of 30, isolation window of 1 m / z, and fragmentation energy of 32.

[0015] In a second aspect, the present application provides a targeted proteomic analysis method, which comprises: obtaining a mass spectrum raw file by subjecting a polypeptide solution obtained by enzymatic digestion of a protein sample to liquid chromatography tandem mass spectrometry analysis; the mass spectrometry acquisition mode is FullMS-PRM, and the mass spectrum raw file comprises a primary full scan mass spectrum and a secondary PRM mass spectrum; based on a proteome database, using a library searching software to analyze the mass spectrum raw file to obtain a library searching result, and selecting a qualitative and / or quantitative result of a target protein from the library searching result;

[0016] The acquisition parameters of the primary full scan mass spectrum comprise:

[0017] (a) positive ion mode, (b) Orbitrap resolution greater than 10000, (c) scan range of 100-2000 m / z, (d) automatic gain control target (AGC target) greater than 8e3, (e) 10-30 ms of precursor ion accumulation time for scanning of the primary mass spectrum;

[0018] The acquisition parameters of the secondary PRM mass spectrum comprise:

[0019] (a) positive ion mode, (b) high-energy induced fragmentation mode for fragmentation, (c) Orbitrap resolution greater than 10000, (d) default charge state greater than 1, (e) 80-150 ms of ion accumulation time, (f) loop count greater than 20 times, (g) isolation window of 1-3 m / z, and (h) fragmentation energy (NCE) of 25-40;

[0020] The primary full scan mass spectrum and the secondary PRM mass spectrum are simultaneously acquired.

[0021] Preferably, the acquisition parameters of the secondary PRM mass spectrum comprise: Orbitrap resolution of 15000, default charge state of 2, 100 ms of ion accumulation time, loop count of 30, isolation window of 1 m / z, and fragmentation energy of 32.

[0022] In an optional embodiment, the acquisition parameters of the mass spectrum comprise: primary full scan resolution of 60000, automatic gain control target (AGC target) of 3e6, 20 ms of precursor ion accumulation time (Max IT), and scan range of 150-2000 m / z;

[0023] Secondary PRM mass spectrum resolution of 15000, automatic gain control target (AGC target) of 1e5, 100 ms of ion accumulation time (Max IT), loop count of 30, isolation window of 1 m / z, and fragmentation energy (NCE) of 32.

[0024] In an alternative embodiment, the search engine is selected from Proteomic Discoverer or MaxQuant.

[0025] In an alternative embodiment, the search engine is Proteomic Discoverer and the search parameters are: peptide tolerance: 10 ppm; MS / MS tolerance 0.02 Da; variable modifications: Oxidation on Methionine / +15.995 Da, Acetyl on N-terminus / +42.011 Da; fixed modification: Carbamidomethyl on Cysteine / +57.021 Da; Enzyme: Trypsin; Miss Cleavage: 2.

[0026] In an alternative embodiment, the search engine is MaxQuant and the search parameters are: Main search peptide tolerance: 4.5 ppm; variable modifications: Acetyl on N-terminus / +42.011 Da; fixed modification: Carbamidomethyl on Cysteine / +57.021 Da; Enzyme: Trypsin; Miss Cleavage: 2.

[0027] In an alternative embodiment, the detection condition of the liquid chromatography is:

[0028] The trapping column and the elution column are both C18.

[0029] The mobile phase A is acetonitrile aqueous solution containing 0.1% (w / v) formic acid, the volume ratio of acetonitrile and water is less than 50%, preferably 2:98.

[0030] The mobile phase B is acetonitrile aqueous solution containing 0.1% (w / v) formic acid, the volume ratio of acetonitrile and water is greater than 50%, preferably 98:2.

[0031] In a third aspect, the present application provides the use of the analysis method of any of the preceding embodiments in any of (a)-(d):

[0032] (a) drug design or screening;

[0033] (b) tissue source identification;

[0034] (c) pathological science research;

[0035] (d) species identification.

[0036] The application provides a targeted proteomics data processing method based on a library searching software, which realizes automatic operation by only importing PRM mass spectrum data and setting library searching parameters, and finally obtains accurate and reliable qualitative and quantitative results by using the library searching software to process the PRM mass spectrum data. The method does not need manual intervention during operation, reduces human error, makes up for the problems in processing targeted proteomics data by Skyline, and the result obtained by automatic processing of the library searching software is also superior to Skyline in accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0038] Figure 1 Skyline analysis PRM data result provided in embodiment 1.4 of the present application;

[0039] Figure 2 Correlation of retention time of target polypeptide in standard 129 sample in three software in embodiment 1.5.1 of the present application;

[0040] Figure 3 DDA quantitative result and PRM quantitative result heat map of target polypeptide in standard 129 sample in embodiment 1.5.2 of the present application;

[0041] Figure 4 Detection result of polypeptide corresponding to protein meeting 2-fold and 9-fold difference in embodiment 1.5.2 of the present application;

[0042] Figure 5 Skyline analysis PRM data result provided in embodiment 2.4.1 of the present application;

[0043] Figure 6 Correlation of retention time of target polypeptide in different tissue samples of mice in embodiment 2.5.1 of the present application;

[0044] Figure 7 DDA quantitative result and PRM quantitative result heat map of target polypeptide in different tissue samples of mice in embodiment 2.5.2 of the present application. DETAILED DESCRIPTION

[0045] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application.

[0046] Term explanation:

[0047] The term "Full MS" refers to full scan of mass to charge ratio.

[0048] The term "AGC target 3e6" refers to automatic gain control target of 3000000 in mass spectrum acquisition process.

[0049] The term "Max IT" refers to maximum injection time.

[0050] The term "loop count" refers to loop count.

[0051] The term "NCE" refers to fragmentation energy.

[0052] The term "isolation window" refers to isolation window for acquiring each spectrum.

[0053] The term "search database" is an indispensable step in conventional shotgun proteomics. Proteins are digested into polypeptides, and then analyzed by mass spectrometry. Mass spectrometry can generate thousands of spectra within an hour, and it is unrealistic to manually compare and analyze these spectra one by one. Therefore, the concept of search database is proposed, and computer software and programs are used to automatically compare and analyze the generated spectra to give the final protein / polypeptide qualitative and quantitative results. Currently, Proteomic Discoverer (PD) and MaxQuant (MQ) are two commonly used search database software in high-throughput proteomics, and their performance has been widely evaluated. The search database software first processes the spectra through polypeptide identification search engines such as Sequest HT and Mascot, which can quickly and accurately identify polypeptides in complex samples. The non-labeling quantification (LFQ) based on the search database software can align the retention time through feature mapping, realize cross-file feature linking, and ensure the consistency of qualitative and quantitative results between samples in the same batch.

[0054] Based on the above advantages of the search library software, the application provides a targeted proteomics data processing method based on search library software. By using conventional search library software, mainly PD and MQ, targeted proteomics data, mainly PRM data, are processed. Based on the powerful performance of the search library software, only the PRM raw data needs to be imported, and the search library parameters are set to automatically run, and finally accurate and reliable qualitative and quantitative results are obtained. The method does not require manual intervention during operation, reduces human error, and makes up for the problems in processing targeted proteomics data by Skyline. And the results obtained by the automatic processing of the search library software are even better than Skyline in terms of accuracy.

[0055] Overall, the search library software analysis method provided by the application comprises:

[0056] (1) The target protein / polypeptide sample is analyzed by liquid chromatography tandem mass spectrometry in Full MS-PRM mode to obtain mass spectrum data;

[0057] (2) The mass spectrum data collected in (1) is analyzed in the proteome search library software using search library software such as PD (Proteomic Discoverer) or MQ (MaxQuant) to obtain search library results;

[0058] (3) The qualitative and quantitative results of the target protein / polypeptide are selected from the search library results obtained in (2).

[0059] In detail, the search library software analysis method provided by the application comprises:

[0060] (1) The protein sample (such as tissue protein extract) is enzymatically purified and then resuspended with 0.1% (w / v) formic acid aqueous solution, and analyzed by liquid chromatography tandem mass spectrometry. 1 μg of polypeptide sample is injected, and the effective gradient time is 0-90 min. The target polypeptide list based on the target protein screening to be studied in targeted proteomics is imported into the mass spectrometry acquisition method setting file, and edited to form a targeted acquisition method. Mass spectrometer type: mass spectrometer containing a quadrupole and a high-resolution detector. Mass spectrometry acquisition mode Full MS-PRM. Full MS resolution 60000, AGC target 3e6, Max IT 20ms, scan range 150-2000m / z; PRM resolution 15000, AGC target 1e5, Max IT 100ms; loop count 30; isolation window 1m / z; NCE 32, and the mass spectrum raw file is collected.

[0061] (2) Using the proteome database of the corresponding source species as the background library, the mass spectrum raw file collected in (1) is analyzed in the proteome search software, and finally the protein / polypeptide qualitative / quantitative result is obtained.

[0062] (3) The target protein / polypeptide is selected from the qualitative / quantitative result file of the protein / polypeptide obtained in (1), that is, the qualitative / quantitative result of the target protein protein / polypeptide is finally obtained.

[0063] Some embodiments of the present application will be described in detail below with reference to the accompanying drawings. The following examples and features in the examples can be combined with each other without conflict.

[0064] Example 1

[0065] 1.1 Preparation of standard sample (named 129 polypeptide):

[0066] Proteins were extracted from 293T cells, Arabidopsis thaliana leaves and Escherichia coli, respectively, and then polypeptide solutions were obtained by protease digestion. The standard 129 polypeptide sample was obtained by mixing according to the following proportions, and the three groups of samples were named according to the content of Escherichia coli.

[0067] Standard 129 polypeptide sample 129_1 129_2 129_9 Escherichia coli 1 μg 2 μg 9 μg Arabidopsis leaf 9 μg 8 μg 1 μg 293T cells 90 μg 90 μg 90 μg

[0068] 1.2 Establishment of PRM analysis method:

[0069] 1.2.1 DDA (Data Dependent Acquisition) analysis of standard 129 polypeptide sample

[0070] Three 129 standard polypeptides were added with equal amount of internal standard polypeptide (2x iRT Kit; manufacturer: Biognosys; product number: Ki-3002-2), mixed and centrifuged to take the supernatant for analysis. The sample was detected by LC-MS / MS system (Thermo Scientific TM EASY-nLC TM 1200; Q Exactive HF-X), each sample was repeated three times, and then the results were analyzed.

[0071] The sample was first added to a C18 capture column (3 pm, 75 pm x 45 mm), and then gradient eluted from a C18 analysis column (1.9 pm, 150 pm x 250 mm) at a flow rate of 600 nL / min. The mobile phases were Buffer A (2% acetonitrile / 0.1% formic acid / 98% H2O, i.e., an acetonitrile aqueous solution containing 0.1% (w / v) formic acid, wherein the volume ratio of acetonitrile and water is 2:98) and Buffer B (98% acetonitrile / 0.1% formic acid / 2% H2O, i.e., an acetonitrile aqueous solution containing 0.1% (w / v) formic acid, wherein the volume ratio of acetonitrile and water is 98:2), respectively, and the chromatographic gradient was 100 min. The mass spectrometry parameters were as follows: acquisition time 90 min, positive ion mode; Orbitrap resolution of primary and secondary acquisition 60000 and 15000, respectively; scan range 350-1500 m / z; quadrupole isolation window 1.6 m / z; and high-energy induced fragmentation (HCD) was used for fragmentation. The primary mass spectrum was scanned with a precursor ion accumulation time of 20 ms, and the secondary mass spectrum of 40 precursor ions was collected with a precursor ion accumulation time of 45 ms. The dynamic exclusion time of the precursor ion was set to 20 s.

[0072] 1.2.2 Screening of differential proteins and constant proteins based on standard 129 polypeptide sample DDA data

[0073] The standard 129 polypeptide DDA offline data was first searched in the database by using PD software, and the search parameters were set as follows: peptide tolerance: 10 ppm; MS / MS tolerance 0.02 Da; variable modification: Oxidation / +15.995 Da (M, on methionine), Acetyl / +42.011 Da (N-terminus); fixed modification: Carbamidomethyl / +57.021 Da (C); Enzyme: Trypsin; Miss Cleavage: 2. Then, differential proteins were screened based on the search results, mainly selecting proteins derived from Escherichia coli, which met the quantitative results of 2-fold and 9-fold difference, and some proteins that were constant in the three groups of 129 polypeptide samples, including internal standard proteins.

[0074] 1.2.3 Establishment of PRM method based on screened differential proteins and constant proteins

[0075] First, the corresponding polypeptides of the screened differential proteins and constant proteins were selected, and the polypeptides must be unique polypeptides in a protein, without missed cleavage and modification, and with high relative abundance. Finally, a list of target polypeptides screened was derived, containing the m / z (mass-to-charge ratio) of the polypeptide, the number of charges, and the retention time (the corresponding acquisition time period in the PRM method was the retention time plus or minus 5 minutes).

[0076] 1.3 Standard 129 peptide samples were subjected to PRM analysis:

[0077] 1.3.1 The processing methods and loading volumes for the three 129 standard peptides are the same as for DDA analysis. The chromatographic methods are also the same as for DDA analysis. Import the PRM method obtained above into the mass spectrometry acquisition method settings file and edit it to form a targeted acquisition method. Mass spectrometry acquisition mode: Full MS-PRM. Full MS resolution: 60000, AGC target: 3e6, Max IT: 20ms, scan range: 150–2000 m / z; PRM resolution: 15000, AGC target: 1e5, Max IT: 100ms; loop count: 30; NCE: 32.

[0078] 1.3.2 The processing is the same as 1.3.1, except that the mass spectrometry acquisition mode has removed Full MS and only PMR is available. The parameter settings are the same as 1.3.1.

[0079] 1.4 Standard 129 Sample PRM Data Processing:

[0080] 1.4.1 Skyline analyzes PRM data

[0081] The DDA search results were used as the spectral library for the Skyline software. The target peptide sequences and PRM data from 129 samples were imported into the Skyline software. Each peptide was quantified based on secondary spectra. The automatic quantification results were manually checked one by one, and the integration boundaries were adjusted to ensure that the integration boundaries for the same target peptide were consistent across all samples. Figure 1 As shown, the purple background represents the automatic integration result, and the double dashed lines indicate the adjusted result. The Skyline automatic integration boundary will vary across different samples (whether small peaks are included) and may even be completely incorrectly integrated, requiring manual adjustment based on retention time. This figure aims to directly demonstrate the differences in the Skyline automatic integration boundary between samples using the software interface, providing a more intuitive view. The text in the interface is not the key content of this figure; those skilled in the art will understand the text content upon opening the software. This figure does not need to clearly show the text content. Finally, the retention time and relative abundance of the target peptide are exported.

[0082] 1.4.2 PD Analysis of PRM Data

[0083] The PRM data of standard 129 sample was imported into PD software, and the library parameter settings were the same as 1.2.2. The library search process was as follows: ① select the spectrum file to be processed and recalibrate the mass of the precursor ion; ② select and search the spectrum for the next step processing; ③ screen the spectrum based on the set standard; ④ use Sequest HT software to search the spectrum to get the polypeptide sequence; ⑤ use INFERYS to record the PMS (Peptide-Spectrum Match, spectrum matched to polypeptide) based on intensity; ⑥ use Percolator to calculate the posterior error probability and q value of PSM; ⑦ according to the specified quantitative method to detect the chromatographic peak and feature. The first six steps are mainly for polypeptide quantification, and ⑧ is the quantitative processing. After the library search is completed, the polypeptide list can be exported, and the retention time and relative abundance of the target polypeptide are screened out.

[0084] 1.4.3 MQ analysis of PRM data

[0085] The PRM data of standard 129 sample was imported into MQ software, and the library parameter settings were as follows: Main search peptide tolerance: 4.5ppm; variable modification: Acetyl / +42.011Da (N-terminal); fixed modification: Carbamidomethyl / +57.021Da (C); Enzyme: Trypsin; Miss Cleavage: 2. The library search process refers to the analysis process of PD software, and the retention time and relative abundance of the target polypeptide are screened out from the polypeptide list after the library search is completed.

[0086] 1.5 Comparison of results of three data processing software:

[0087] The results show that the PRM data obtained by the mass spectrum acquisition mode of only PMR without Full MS cannot be processed by the library software. The following results are obtained from the mass spectrum acquisition mode of Full MS-PMR.

[0088] 1.5.1 Comparison of qualitative results

[0089] The library software performs qualitative analysis by spectrum matching, and then the qualitative results are verified again, and finally relatively accurate qualitative results are obtained. However, the target peak selected by Skyline may be incorrect. The target polypeptide selected in standard 129 sample has good peak correlation in three software, and the retention time obtained is relatively consistent, that is, the qualitative results are consistent.

[0090] 1.5.2 Comparison of quantitative results

[0091] The relative quantification results of the target peptide obtained from the three software programs were compared with the original quantification results of DDA. For example... Figure 2 As shown, the differential protein quantification results obtained from the three software programs correlated well with the original DDA quantification results. Only the MQ search result showed a lower correlation due to a large number of missing values. Furthermore, the heatmap ( Figure 3 As can be seen from the results, the constant peptides screened from DDA also remain relatively constant in the PRM results after processing with the three software programs. In summary, this demonstrates that the relative quantification results obtained from processing PRM data with the three software programs are in good agreement with the original DDA results.

[0092] Figure 3 The amino acid sequences (SEQ ID No: 1-52) of the target polypeptide are shown below:

[0093]

[0094]

[0095] The peptides corresponding to proteins that show differences of 2-fold and 9-fold in the standard 129-fold sample are compared, based on DDA and PRM results, to establish an actual fold relationship. For example... Figure 4 As shown in A, the differentially expressed peptides generally conform to a 2-fold relationship, with the PRM data processed by MQ being closest to 2-fold; the PRM data processed by PD showed the smallest dispersion in conforming to the 2-fold relationship. Figure 4 In the data analysis, the multiples of PRM data processed by Skyline are significantly higher than the actual values ​​(average close to 11 times), and the dispersion is also relatively large. The multiples of PRM data processed by the database search software PD and MQ are both close to the actual values ​​(around 9 times), and the data clustering is also relatively high. In conclusion, this suggests that the relative quantitative results obtained from processing and analyzing PRM data using database search software are likely more accurate and reliable than those obtained by Skyline.

[0096] Experimental Example 2

[0097] 2.1 Preparation of different tissue samples from mice:

[0098] Referring to the protein extraction method in the existing technology (DOI: 10.1016 / j.jprot.2021.104456), three tissues of mice, namely liver, brain and kidney, were extracted for protein extraction. After enzymatic hydrolysis and desalting purification, the corresponding peptides of the three tissues were obtained. After freeze-drying, they were reconstituted with 0.1% formic acid for quantification.

[0099] 2.2 Establishment of PRM analysis method:

[0100] 2.2.1 DDA (Data Dependent Acquisition) analysis of polypeptides from different tissues of mice

[0101] Polypeptides from three different tissues were added with equal amount of internal standard polypeptides (2x iRT Kit; Vendor: Biognosys; Product No: Ki-3002-2), mixed well and centrifuged to take supernatant for analysis. The samples were detected by LC-MS / MS system (Thermo Scientific TM EASY-nLC TM 1200; Q Exactive HF-X), and then the results were analyzed. The samples were first loaded onto a C18 trap column (3 pm, 75 pm x 45 mm), and then eluted from a C18 analytical column (1.9 pm, 150 pm x 250 mm) at a flow rate of 600 nL / min. The mobile phases were Buffer A (2% acetonitrile / 0.1% formic acid / 98% H2O) and Buffer B (98% acetonitrile / 0.1% formic acid / 2% H2O), respectively, and the chromatographic gradient was 100 min. The mass spectrometry parameters were as follows: acquisition time 90 min, positive ion mode; Orbitrap resolution of primary and secondary acquisition 60000 and 15000, respectively; scan range 350-1500 m / z; quadrupole isolation window 1.6 m / z; and high-energy induced dissociation (HCD) mode for dissociation. The ion accumulation time was 20 ms for primary mass spectrum scanning, and the ion accumulation time was 45 ms for secondary mass spectrum scanning of 40 precursor ions. The dynamic exclusion time of precursor ions was set to 20 s.

[0102] 2.2.2 Screening of differential proteins and constant proteins based on DDA data of three different tissue samples

[0103] The DDA data of polypeptides from three tissues of mice were first searched by PD software, and the search parameters were set as follows: peptide tolerance: 10 ppm; MS / MS tolerance 0.02 Da; variable modification: Oxidation / +15.995 Da (M), Acetyl / +42.011 Da (N-terminus); fixed modification: Carbamidomethyl / +57.021 Da (C); Enzyme: Trypsin; Miss Cleavage: 2. Then, differential proteins were screened based on the search results, and proteins with different fold changes were selected, and some proteins that were relatively constant in the three tissues were also selected, including internal standard polypeptides.

[0104] 2.2.3 Establishment of PRM method based on screened differential proteins and constant proteins

[0105] Firstly, based on the screened differential proteins and constant proteins, the corresponding polypeptides are selected. The polypeptides must be unique polypeptides of the proteins, and there is no missed cutting and no modification, and the relative abundance is high. Finally, the target polypeptide list screened is derived, including the m / z (mass-to-charge ratio) of the polypeptide, the charge number, and the retention time (the corresponding acquisition time period in the PRM method is the retention time ± 5 minutes).

[0106] 2.3 PRM analysis of polypeptide samples in different tissues of mice

[0107] The polypeptide treatment method and the loading volume of the mice in different tissues are the same as those in the DDA analysis. The chromatographic method is also the same as that in the DDA analysis. The PRM method obtained above is introduced into the mass spectrometry acquisition method setting file, and edited to form a targeted acquisition method. The mass spectrometry acquisition mode is Full MS-PRM. The Full MS resolution is 60000, the AGC target is 3e6, the Max IT is 20 ms, and the scan range is 150-2000 m / z; the PRM resolution is 15000, the AGC target is 1e5, the Max IT is 100 ms; the loop count is 30; the isolation window is 1 m / z; and the NCE is 32.

[0108] 2.4 PRM data processing of three tissue samples of mice

[0109] 2.4.1 Skyline analysis of PRM data

[0110] The DDA search library result is used as the spectrum library of the Skyline software, the target polypeptide sequence and the PRM off-line data of the three tissue samples of mice are imported into the Skyline software, the secondary spectrum is used for quantification of each peptide segment, the automatic quantification result is checked manually one by one, the integration boundary is adjusted, and it is tried to ensure that the integration boundary of the same target polypeptide is consistent in all samples. As shown in A of Figure 5 , the purple background is the automatic integration result, the green frame is the correct peak, and the red frame is the automatic integration error peak. When the target polypeptide peak has a relatively low relative abundance in the acquisition time period, the automatic integration is prone to error, and the peak with a relatively high relative abundance will be incorrectly selected. As shown in B of Figure 5As shown in Figure B, when the target peptide peak is not fully acquired (due to incorrect acquisition window settings), Skyline's automatic integration will also produce errors, requiring manual adjustment. However, this cannot compensate for the low peak area caused by incomplete peak acquisition. The purpose of this attached figure is to directly demonstrate the differences in the Skyline automatic integration boundary between samples using the software interface, providing a more intuitive view. The text in the interface is not the key content of this attached figure; those skilled in the art will clearly understand the text content after opening the software. This attached figure does not need to clearly display the text content. After manual detection, the retention time and relative abundance of the target peptide are exported.

[0111] 2.4.2 PD Analysis of PRM Data

[0112] Import the PRM data from different mouse tissue samples into the PD software, setting the search parameters as in section 2.2.2. After the search is complete, a peptide list can be exported, from which the retention time and relative abundance of the target peptide can be selected.

[0113] 2.4.3 MQ Analysis of PRM Data

[0114] PRM data from different mouse tissue samples were imported into the MQ software. The search parameters were set as follows: Mainsearch peptide tolerance: 4.5 ppm; Variable modification: Acetyl / +42.011 Da (N-terminus); Fixed modification: Carbamidomethyl / +57.021 Da (C); Enzyme: Trypsin; Miss Cleavage: 2. After the search, the retention time and relative abundance of target peptides were selected from the peptide list.

[0115] 2.5 Comparison of results from three data processing software programs:

[0116] 2.5.1 Comparison of Qualitative Results

[0117] The database search software performs qualitative analysis by comparing spectra, and then verifies the qualitative results again to obtain a relatively accurate qualitative result. However, Skyline's automatic integration may result in errors in the selected target peak. Figure 5 The target peptides selected from three mouse tissue samples showed good peak correlation in the three software programs. Figure 6 The retention times obtained are relatively consistent, that is, the qualitative results are consistent.

[0118] 2.5.2 Comparison of Quantitative Results

[0119] The relative quantification results of the target peptide obtained from the three software programs were compared with the original quantification results of DDA. For example... Figure 7As shown, the three software-derived differential protein quantitative results are well correlated with the DDA original quantitative results. In addition, it can also be seen from the heat map that the constant polypeptides screened in DDA remain relatively constant in the PRM results after processing by the three software. In summary, it can be shown that the relative quantitative results obtained by processing PRM data by the three software are well consistent with the original DDA results. In addition, there are three target polypeptides (ILEATAHAQAQLGCPVIIHPGR, ILEATAHAQAQLGCPVIIHPGR, VAHSFNCTPIEGMLSHQLK) whose mass spectrometry acquisition window settings are wrong, resulting in incomplete polypeptide acquisition. After manual adjustment of integration in Skyline, the peak area cannot be completed, resulting in differences within the group. However, the search software can compare the peak areas according to the sample, complete the peak area of the target polypeptide that has not been acquired completely, and thus obtain more accurate results.

[0120] Figure 7 The amino acid sequences of the target polypeptides in the above-mentioned polypeptide library (SEQ ID No: 53-113) are as follows:

[0121]

[0122] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A targeted proteomics analysis method, characterized in that, The analytical method includes obtaining a raw mass spectrum file by liquid chromatography-tandem mass spectrometry analysis of the polypeptide solution after enzymatic digestion of the protein sample; the mass spectrometry acquisition mode is Full MS-PRM, and the raw mass spectrum file includes a first-level full-scan mass spectrum and a second-level PRM mass spectrum. Based on a proteome database, the mass spectrum raw file was analyzed using a search software to obtain the search results, and the qualitative and / or quantitative results of the target protein were selected from the search results. The parameters for acquiring the first-level full-scan mass spectrum include: (a) In positive ion mode, (b) Orbitrap resolution greater than 10000, (c) scan range of 100~2000 m / z, (d) automatic gain control target greater than 8e3; (e) scan of primary mass spectra with precursor ion accumulation time of 10~30 ms. The parameters for obtaining the secondary PRM mass spectrum include: (a) In positive ion mode, high-energy induced fragmentation is used for fragmentation; (b) Orbitrap resolution is greater than 10000; (c) default charge state is greater than 1; (d) ion accumulation time is 80~150ms; (e) number of cycles is greater than 20; (f) isolation window is 1~3m / z; (g) fragmentation energy is 25~40. The first-level full-scan mass spectrum and the second-level PRM mass spectrum were acquired simultaneously. The database search software is selected from Proteomic Discoverer or MaxQuant; The search parameters for the Proteomic Discoverer search software are as follows: Peptide tolerance: 10 ppm; MS / MS tolerance: 0.02 Da; Variable modifications: Oxidation on methionine / +15.995 Da, Acetyl at the N-terminus / +42.011 Da; Fixed modification: Carbamidomethyl on cysteine / +57.021 Da; Enzyme: Trypsin; Miss Cleavage: 2; The search parameters for the MaxQuant database search software are as follows: Main search peptide tolerance: 4.5 ppm; Variable modification: Acetyl at the N-terminus / +42.011 Da; Fixed modification: Carbamidomethyl on cysteine / +57.021 Da; Enzyme: Trypsin; Miss Cleavage:

2.

2. The analytical method according to claim 1, characterized in that, The parameters for acquiring the secondary PRM mass spectrum include: Orbitrap resolution of 15000, default charge state 2, ion accumulation time of 100 ms, number of cycles of 30, isolation window of 1 m / z, and fragmentation energy of 32.

3. The analytical method according to claim 1, characterized in that, The mass spectrum acquisition parameters include: The resolution of the first-level full scan is 60,000, the target of automatic gain control is 3e6, the precursor ion accumulation time is 20ms, and the scan range is 150~2000 m / z. The resolution of the secondary PRM mass spectrometry scan was 15000, the automatic gain control target was 1e5, the precursor ion accumulation time was 100ms, the number of cycles was 30, the isolation window was 1 m / z, and the fragmentation energy was 32.

4. The analytical method according to claim 1, characterized in that, The detection conditions for the liquid chromatography are as follows: Both the capture column and the elution column are C18; Mobile phase A is an aqueous solution of acetonitrile containing 0.1% (w / v) formic acid, with the volume ratio of acetonitrile to water being less than 50%. Mobile phase B is an aqueous solution of acetonitrile containing 0.1% (w / v) formic acid, with a volume ratio of acetonitrile to water greater than 50%.

5. The application of the analytical method according to any one of claims 1 to 4 in any one of (a) to (d): (a) Drug design or screening; (b) Identification of the organization's origin; (c) Pathological scientific research; (d) Species identification.

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