A high-throughput method for analyzing intact s-palmitoylated modified proteins / peptides and applications thereof

Abstract

The application belongs to the technical field of biochemical analysis, and relates to a method for high-throughput analysis of complete S-palmitoylation modified proteome in organisms and application thereof. The application uses fluorinated graphite material as a solid phase carrier, establishes a selective separation method for complete S-palmitoylation modified peptide segments in biological samples through twice enrichment strategies, realizes combined sample dissolution analysis by changing the acetonitrile concentration in the sample dissolution solution, improves the recovery rate of S-palmitoylation modified peptide segments when the chromatographic sample is fed, and establishes a high-throughput mass spectrometric analysis method for complete S-palmitoylation modified peptide segments through an optimized chromatographic elution gradient and the setting of database search parameters. The method can analyze and identify S-palmitoylation modified proteins in biological samples on a large scale, and simultaneously obtains information about modification sites and modification groups. The method has high reliability and provides rich information.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemical analysis, and particularly relates to a method for high-throughput analysis of intact S-palmitoylation modified proteins / peptides BACKGROUND

[0002] The prior art discloses that S-palmitoylation modification (accurately referred to as S-acylation modification) is a dynamic and reversible post-translational modification form of proteins, which regulates the subcellular localization, activity, stability, protein-protein interaction, etc. of proteins in vivo, and participates in a series of important physiological and pathological processes such as signal transduction, occurrence and development of diseases (such as cancer), etc.

[0003] The prior art mainly has two types for enrichment of the S-palmitoylation modification group of proteins: a “palmitic acid”-centered method based on palmitic acid analog metabolic labeling and click chemistry, and a “cysteine”-centered method based on neutral hydroxylamine hydrolysis and thiol-specific reaction. However, the two types of methods have their own shortcomings, such as the “palmitic acid”-centered method cannot be used for analyzing tissue and body fluid samples, and the stimulation of exogenous introduction of high-concentration palmitic acid analogs may cause unpredictable biological effects, and the analysis ability is different for modified proteins with different update rates; the “cysteine”-centered method belongs to an indirect detection method, and has a high false positive rate; and the two types of methods cannot realize high-throughput analysis of intact S-palmitoylation modified peptides (containing site and modification group information).

[0004] In view of the problems in the prior art analysis technology, the inventors of the present application propose to realize selective separation of intact S-palmitoylation modified peptides in biological samples based on fluorinated graphite material and twice enrichment strategy, and to establish a high-throughput mass spectrometry analysis method for intact S-palmitoylation modified peptides by means of a step-by-step sample dissolution method and improved database search parameter setting. SUMMARY

[0005] The purpose of the present application is to provide a method for selective separation and enrichment of intact S-palmitoylation modified peptides and high-throughput mass spectrometry analysis in view of the problems in the prior art analysis technology. Specifically, the present application relates to a method for high-throughput analysis of intact S-palmitoylation modified proteins / peptides and application thereof.

[0006] The application provides a method for separating and purifying intact S-palmitoylated modified peptide segments in a biological sample based on a fluorinated graphite material solid-phase extraction technique; a method for optimizing the concentration of acetonitrile in a sample dissolution solution, taking into account the dissolution efficiency and recovery rate of S-palmitoylated modified peptide segments during chromatographic sampling; and a method for establishing a high-throughput mass spectrometry identification and analysis method for intact S-palmitoylated modified peptide segments by optimizing the chromatographic elution gradient and database search parameter settings. The method is used for the analysis of actual biological samples.

[0007] Specifically, the application verifies the feasibility of using fluorinated graphite material (purchased from Jiangsu Xianfeng Nanometer Material Technology Co., Ltd.) as a solid-phase carrier to selectively extract and separate intact S-palmitoylated modified peptide segments. Experiments prove that, compared with non-palmitoylated modified peptides with the same amino acid sequence, the fluorinated graphite material has good selective adsorption effect on S-palmitoylated modified peptide segments, and has obvious enrichment effect on modified peptide segments in a complex mixed system (the molar ratio of modified peptide segments to MYO doped non-modified peptide segments is 1:100).

[0008] The application discloses a method for selectively separating and enriching intact S-palmitoylated modified peptide segments based on twice fluorinated graphite material solid-phase extraction. The separation effect of the twice enrichment method on S-palmitoylated modified standard peptide segments is tested by doping BSA enzymatic peptide segment mixture. The incubation system of the sample to be separated and the fluorinated graphite material is 50% methanol solution; in order to remove non-specific adsorption on the fluorinated graphite material, the washing solution used is 20% acetonitrile, 50% methanol and 60% methanol; and the peptide segment elution solution is 90% methanol and 100% methanol. After the first enrichment of the peptide segment mixture is frozen and redissolved in 50% methanol solution, the washing solution used is 50% methanol and 60% methanol, and the peptide segment elution process remains unchanged, and the second enrichment is performed. Experiments show that, compared with the one-time enrichment method, the twice enrichment method can better separate and purify S-palmitoylated modified peptide segments from a complex sample system.

[0009] The application provides a method for combining samples with different acetonitrile concentrations to improve the dissolution efficiency and recovery rate of S-palmitoylated modified peptide segments during chromatographic sampling. The improved sample dissolution solution is 15% ACN+0.1% FA, 35% ACN+0.1% FA and 55% ACN+0.1% FA. Experiments show that the three sample dissolution methods have good complementarity, and the combined use can improve the identification efficiency of S-palmitoylated modified peptide segments.

[0010] The application provides a gradient setting for elution when chromatographic separation is performed by using the different sample dissolving manners: the chromatographic gradient of the sample dissolved by 15% ACN+0.1% FA is 0min-30min, 15%-40% B phase, 30min-35min, 40%-95% B phase, 35min-45min, 95% B phase; the chromatographic gradient of the sample dissolved by 35% ACN+0.1% FA is 0min-30min, 35%-65% B phase, 30min-35min, 65%-95% B phase, 35min-45min, 95% B phase; and the chromatographic gradient of the sample dissolved by 55% ACN+0.1% FA is 0min-25min, 55%-85% B phase, 25min-30min, 85%-95% B phase, 30min-35min, 95% B phase. The mobile phase A is 100% H2O containing 0.1% FA, and the mobile phase B is 100% ACN containing 0.1% FA. Meanwhile, the experimental results show that, in the CID mass spectrum fragmentation mode, the palmitoyl group remains and loses in the fragment ions generated by the S-palmitoylation modified peptide, and the fragment ions losing the palmitoyl group can be observed in the spectrum. Therefore, in the database retrieval in the application, the Palmitoylation (C) is set as a variable modification, and the sub-ion losing the palmitoyl group is allowed to exist. 16 H 30 O, 238.23 Da).

[0011] The HeLa cell protein is used as a complex biological sample to perform protein enzymolysis, S-palmitoylation modified peptide enrichment, chromatographic separation and mass spectrum analysis, so as to test the feasibility of the method for actual sample analysis.

[0012] The results show that, by improving the gradient setting in the chromatographic separation process and the database retrieval parameter setting in the mass spectrum analysis, the identification scale of the S-palmitoylation modified peptide can be improved, the complete S-palmitoylation modified peptide in the actual biological sample (such as HeLa cell) can be analyzed, and the information of the rich modified sites and the modification groups can be obtained at the same time.

[0013] In the present application, 701 S-palmitoylation modified peptide segments are identified from a HeLa cell sample by the method, corresponding to 714 palmitoylation modification sites, including some well-known palmitoylation modified proteins and sites, and the protein lipid modification crosstalk is found by the opensearch database retrieval mode, that is, different fatty acid (such as C16:0, C16:1) modification states may exist in the same protein or even the same site of the protein. Compared with the existing analysis method, the method of the present application can obtain the information of the modification site and the modification group at the same time through one mass spectrometry, not only improves the reliability of the mass spectrometry identification of S-palmitoylation modified proteins and sites, but also provides more abundant information for the structure and function research of S-palmitoylation modified proteins.

[0014] The present application has the following advantages and beneficial effects:

[0015] The present application provides an analysis method for complete S-palmitoylation modified peptide segment groups based on fluorinated graphite material solid phase extraction. Compared with the prior art, the present method is suitable for the analysis of complete S-palmitoylation modified peptide segments in various samples (tissues, cells and body fluids), and the method is simple and easy to operate; the information of the modification site and the modification group can be obtained at the same time through one mass spectrometry analysis, which improves the identification reliability of the modified peptide segments and sites, and can be further used for the research of lipid modification crosstalk. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Comparison of adsorption effects of fluorinated graphite material on S-palmitoylation modified standard peptide segments (GDFC palm IQVGR) and its control (GDFCIQVGR), wherein,

[0017] a, mass spectrometry diagrams of the supernatant (flow through) before (upper diagram) and after (lower diagram) enrichment of the mixture of GDFC palm IQVGR, GDFCIQVGR and MYO enzymolysis peptide segments; b, quantitative comparison of adsorption effects, n = 3.

[0018] Figure 2 Comparison of washing effects of different solvents on non-specific adsorption peptide segments on fluorinated graphite material, wherein, after the mixture of S-palmitoylation modified peptide segments and MYO enzymolysis peptide segments is incubated with fluorinated graphite material, 20% CH3OH, 40% CH3OH, 60% CH3OH, 70% CH3OH, 20% ACN and 30% ACN are used for washing in sequence, the solutions after each washing are collected, and MALDI MS analysis is performed; * represents the signal of S-palmitoylation modified peptide segments.

[0019] Figure 3 The enrichment effects of the first and second extractions were analyzed. The S-palmitoylated peptide and the BSA-digested peptide were mixed at a molar ratio of 1:500 and subjected to the first and second solid-phase extractions using fluorinated graphite material. The peptide eluates from both extractions were collected and analyzed by MALDI MS. * indicates the signal of the S-palmitoylated peptide.

[0020] Figure 4 Experimental flowchart for solid-phase extraction separation and analysis of complete S-palmitoylated modified peptides based on fluorinated graphite materials.

[0021] Figure 5 The effects of stepwise dissolution of the test samples with acetonitrile solutions of different concentrations were analyzed. a) Identification of S-palmitoylated modified peptides in samples dissolved with 15% ACN + 0.1% FA, 35% ACN + 0.1% FA and 55% ACN + 0.1% FA; b) Coverage of S-palmitoylated modified peptides identified in the three sample dissolution methods.

[0022] Figure 6 Examples of well-known S-palmitoylation modification sites and their mass spectra.

[0023] Figure 7 Examples of new S-palmitoylation modification sites and their mass spectra in the identification results.

[0024] Figure 8 The identification results did not show palmitoyl loss (C). 16 H 30 Example of a mass spectrum (O, 238.23 Da).

[0025] Figure 9 The identification results showed significant palmitoyl loss (C). 16 H 30 Example of a mass spectrum (O, 238.23 Da).

[0026] Figure 10 The identification results show sites with fatty acid modification crosstalk and their mass spectra [Q8NBI5,S43A3_Human site=226], that is, the same site of the protein is modified by different fatty acids (such as C16:0, C16:1) at the same time. Detailed Implementation

[0027] The S-palmitoylated modified standard peptide (GDFC) used in the embodiments of the present invention palmIQVGR; ≥95%) were synthesized by SynPeptide Co., Ltd., China, and the non-palmitoylated standard peptide segment (GDFCIQVGR; ≥98%) was synthesized by ChinaPeptides Co., Ltd. The fluorinated graphite material (nGF) was purchased from Jiangsu Xianfeng Nanometer Material Technology Co., Ltd. and washed with methanol solution before use. Myoglobin (MYO) and bovine serum albumin (BSA) were trypsinized according to methods known in the art, and the peptide segment mixtures obtained by enzymolysis were desalted using a Sep-Pak C18 column according to methods known in the art. HeLa cells were cultured according to methods known in the art.

[0028] Example 1 Investigation of the enrichment selectivity of fluorinated graphite material (nGF) for standard S-palmitoylated modified peptide segments

[0029] (1) The standard S-palmitoylated modified peptide segment (GDFCIQVGR; ≥95%) was dissolved in DMSO to prepare a stock solution with a final concentration of 1 μg / μL, which was used for subsequent experiments. palm IQVGR) was dissolved in DMSO to prepare a stock solution with a final concentration of 1 μg / μL, which was used for subsequent experiments.

[0030] (2) The S-palmitoylated modified standard peptide segment, the non-modified standard peptide segment and the MYO enzymolysis peptide segment were mixed in a molar ratio of 1:1:2, vacuum freeze-dried, and then completely dissolved in 50% methanol aqueous solution. nGF material was added to the above peptide segment mixture in an amount corresponding to 1 mg of nGF per 250 ng of S-palmitoylated modified peptide segment, and the reaction was incubated at room temperature for 2 h. After centrifugation at 11000 rcf for 10 min, the supernatant was collected and freeze-dried.

[0031] (3) The modified standard peptide segment + non-modified standard peptide segment + MYO peptide segment mixture and the peptide segments collected from the supernatant in (2) were detected by MALDI-TOF mass spectrometry (5800 MALDI-TOF / TOF-MS) according to methods known in the art.

[0032] (4) The R I value was introduced and calculated. I The larger the R I value, the stronger the binding of the peptide segment to nGF, i.e., the stronger the interaction. In the formula, I represents the relative intensity of a specific peptide segment to a reference peptide segment, and the subscripts initial and flow represent the initial peptide mixture before enrichment (initial mixture) and the supernatant after enrichment (flow through). The reference peptide segment is the MYO peptide segment VEADIAGHGQEVLIR, which has the strongest peak in the spectra of the initial mixture and the flow through.

[0033] R I =(I initial -I flow ) / I initial

[0034] As Figure 1 shown, S-palmitoylation modified peptide segment has greater R I value, i.e. has stronger interaction with nGF under the same incubation condition, thus, S-palmitoylation modified peptide segment can be separated and purified with nGF as enrichment carrier.

[0035] Example 2 Selection of washing solution in separating S-palmitoylation modified peptide segment with nGF solid phase extraction method

[0036] (1) S-palmitoylation modified standard peptide segment was mixed with MYO enzymatic peptide segment at a molar ratio of 1:4, freeze-dried, and then completely dissolved in 50% methanol aqueous solution, nGF material was added, and the reaction was incubated at room temperature for 2h, then centrifuged at 11000rcf for 10min, and the supernatant was discarded;

[0037] (2) 20% CH3OH, 40% CH3OH, 60% CH3OH, 70% CH3OH, 20% ACN and 30% ACN were added as washing solutions respectively, shaken and incubated for 30min, centrifuged, and the supernatant of each washing solution was collected and freeze-dried;

[0038] (3) The supernatant of (2) was detected by MALDI-TOF mass spectrometry;

[0039] As Figure 2 shown, no signal of S-palmitoylation modified standard peptide segment was detected in the collected supernatant when co-incubated and washed with 20% CH3OH, 40% CH3OH, 60% CH3OH and 20% ACN, so they can be used as washing solutions to wash off non-modified peptide segments on the surface of nGF material, while S-palmitoylation modified peptide segments still adsorb on the surface of nGF material.

[0040] Example 3 Effect analysis of separating and purifying S-palmitoylation modified peptide segment by twice nGF solid phase extraction method

[0041] (1) S-palmitoylation modified standard peptide segment was mixed with BSA enzymatic peptide segment at a molar ratio of 1:500, freeze-dried, and then completely dissolved in 50% methanol aqueous solution, nGF material was added, and the reaction was incubated at room temperature for 2h, then centrifuged at 11000rcf for 10min, and the supernatant was discarded;

[0042] (2) 20% ACN, 50% CH3OH and 60% CH3OH were added as washing solutions in turn, shaken and incubated for 30min, centrifuged, and the supernatant was discarded;

[0043] (3) Sequentially add 90% CH3OH and 100% CH3OH, shake constantly, incubate at room temperature for 1 h, elute the adsorbed peptide fragments on nGF, combine the eluents and freeze-dry. The above is the first enrichment;

[0044] (4) Redissolve the peptide fragments obtained in (3) in 50% methanol aqueous solution, add nGF material again, and perform the second enrichment according to (1)-(3). The washing solution is 50% CH3OH and 60% CH3OH.

[0045] (5) Collect the peptide fragment samples obtained in (3) and (4) and perform MALDI-TOF mass spectrometry analysis.

[0046] As shown in Figure 3 , after the second enrichment, the signal of the S-palmitoylation modified peptide fragment is the strongest in the mass spectrum, and the signal of the BSA (non-palmitoylation modified protein) peptide fragment is basically not detected, indicating that the separation effect of nGF on the S-palmitoylation modified peptide fragment can be significantly improved after the second enrichment.

[0047] Example 4 Analysis of the complete S-palmitoylation modified peptide fragment group in the HeLa cell sample

[0048] The operation procedure is shown in Figure 4

[0049] (1) HeLa cell protein extraction: The collected HeLa cells were washed twice with pre-cooled PBS solution, and a lysis solution (4% SDS, 150 mM NaCl, 50 mM Tris, 5 mM EDTA, 1x protease inhibitor cocktail and 1 mM PMSF, pH 7.4) was added for protein extraction by ice water bath ultrasonic lysis. The obtained protein lysis product was centrifuged at 16 000 rcf for 30 min at 4°C, and the supernatant was precipitated by the methanol-chloroform precipitation method known in the art;

[0050] (2) Redissolve the sample by adding 4% SDS, 50 mM Tris, 5 mM EDTA, pH 7.4, and perform protein quantification by the BCA method known in the art using ultraviolet-visible spectrophotometry;

[0051] ​(3) The sample obtained in (2) was diluted with 150 mM NaCl, 50 mM Tris, 5 mM EDTA, pH 7.4; tris(2-carboxyethyl)phosphine (TCEP) was added to a final concentration of 10 mM, and the sample was reacted at room temperature for 30 min to reduce and open the disulfide bonds in the protein, and then N-ethylmaleimide (NEM) was added to a final concentration of 50 mM, and the sample was reacted at room temperature in the dark for 1 h to block the free sulfhydryl groups in the protein; the protein sample was precipitated with methanol-chloroform to remove TCEP, NEM and SDS and the like from the sample;

[0052] (4) The protein sample was resolubilized by adding 50 mM Tris-HCl (pH 7.4) and was added with mass spectrometry grade trypsin at a ratio of 1:50 (w / w, enzyme: protein), and was enzymatically digested at 37°C overnight according to the known method for protein enzymatic digestion, and was desalted with a Sep-Pak C18 column, and the peptides were eluted with 70% ACN containing 0.1% TFA, and were concentrated by vacuum freeze-drying;

[0053] (5) The peptide sample obtained in (4) was completely dissolved in an aqueous methanol solution with a final concentration of 50%, and an appropriate amount of nGF material was added, and the sample was incubated at room temperature for 2 h, and was centrifuged at 11000 rcf for 10 min, and the supernatant was discarded;

[0054] (6) 20% ACN, 50% CH3OH and 60% CH3OH were sequentially added as washing solutions, and the sample was incubated by shaking for 30 min, and was centrifuged, and the supernatant was discarded;

[0055] (7) 90% CH3OH and 100% CH3OH were sequentially added, and the sample was incubated at room temperature by constant shaking for 1 h to elute the peptides adsorbed on the nGF, and the eluate was collected by centrifugation, and was freeze-dried;

[0056] (8) The peptides obtained in (7) were resolubilized in an aqueous methanol solution with a concentration of 50%, and an appropriate amount of nGF material was added again, and the second enrichment was performed according to (5) to (7);

[0057] (9) Collect the peptide sample obtained in (8), and dissolve the peptide sample in three different ways, a, divide the sample into three parts, and dissolve each part in 15% ACN + 0.1% FA, 35% ACN + 0.1% FA and 55% ACN + 0.1% FA respectively, and dissolve for 2 hours after ultrasonic dissolution in an ice water bath and oscillation at room temperature; b, dissolve the sample in 1x Invitrosol, and oscillate at room temperature for 2 hours; c, dissolve the sample in 1x Invitrosol for 1 hour at room temperature, then dilute to a final concentration of 0.25x Invitrosol by adding water, and oscillate at room temperature for 1 hour; the sample loading volume and sample dissolution final concentration are respectively: 3 μL and 67 ng / μL (15% ACN + 0.1% FA), 2 μL and 100 ng / μL (35% ACN + 0.1% FA and 55% ACN + 0.1% FA), 1 μL and 200 ng / μL (1x Invitrosol), and 4 μL and 50 ng / μL (0.25x Invitrosol);

[0058] (10) Chromatographic separation and mass spectrometric analysis: In this embodiment, a nano-UHPLC chromatographic system (AURORA Series column, 75 μm i.d. x 25 cm, C18, 1.6 μm) is used, and the flow rate is 300 nL / min. Different sample dissolution methods use different chromatographic gradient settings; the chromatographic gradient is as follows: for the sample dissolved in 15% ACN + 0.1% FA, 0-30 min, 15%-40% B phase, 30-35 min, 40%-95% B phase, 35-45 min, 95% B phase; for the sample dissolved in 35% ACN + 0.1% FA, 0-30 min, 35%-65% B phase, 30-35 min, 65%-95% B phase, 35-45 min, 95% B phase; for the sample dissolved in 55% ACN + 0.1% FA, 0-25 min, 55%-85% B phase, 25-30 min, 85%-95% B phase, 30-35 min, 95% B phase; for the sample dissolved in 1x Invitrosol and 0.25x Invitrosol, the corresponding chromatographic gradient is 0-10 min, 5%-10% B phase, 10-15 min, 10%-30% B phase, 15-60 min, 30%-60% B phase, 60-61 min, 60%-95% B phase, 61-70 min, 95% B phase, and the mobile phase A is 100% H2O containing 0.1% FA, and the mobile phase B is 100% ACN containing 0.1% FA. The flow rate is 300 nL / min. TIMS-TOF Pro mass spectrometer is used for mass spectrometric analysis;

[0059] (11) Database Search: Two database search methods were used on PEAKS Online (https: / / www.bioinfor.com / , Bioinformatics Solutions Inc, PEAKS Online X build). The database used was the Human UniProtKB / Swiss-Prot database (downloaded in July 2021, Homo sapiens, 20381 entries); Restricted search parameters were set as follows: trypsin digestion, maximum of 3 missed cleavage sites; precursor ion error 15 ppm, ion ion error 0.05 Da; Nethylmaleimide (C), Oxidation (M), and Palmitoylation (C) were variable modifications, with Palmitoylation (C) allowing palmoyl loss (C). 16 H 30 O, 238.23 Da); FDR of protein and spectrum matching rate ≤ 1%. Open search was performed using PEAKS PTM with the following parameters: De Novo ALC (%) > 15 (software default parameter), searching all built-in protein post-translational modifications, and other parameter settings were the same as for Restricted search. Peptide score (-10 LgP) and AS score can be used to evaluate the identification results;

[0060] like Figure 5 As shown, the S-palmitoylated modified peptides / proteins identified by using 15% ACN + 0.1% FA, 35% ACN + 0.1% FA, and 55% ACN + 0.1% FA as sample dissolution solutions before chromatographic injection exhibit good complementarity, indicating that different concentrations of acetonitrile solutions have their own emphases on peptide solubility and chromatographic loading efficiency. Therefore, in this embodiment, 15% ACN + 0.1% FA, 35% ACN + 0.1% FA, and 55% ACN + 0.1% FA are combined to improve the recovery rate of the sample before chromatographic injection by using a combined sample dissolution method.

[0061] Using the method of this invention, the inventors identified 701 S-palmitoylated modified peptides corresponding to 714 palmitoylation modification sites in HeLa cell samples, including some well-known palmitoylated modified proteins and sites (such as...). Figure 6 (as shown), and also identified many new modified proteins and sites (such as...) Figure 7 (as shown); Analysis of these omics data revealed that fatty acid modification groups in S-palmitoylated peptides may be retained on the peptide chain during CID mass spectrometry fragmentation (e.g.Figure 8 It is possible that there is no palmitoyl loss (as shown), but it is also possible that there is a slight or significant palmitoyl loss (as shown). Figure 9 Therefore, palmitoylation (C) is set as a variable modification, and the presence of palmitoyl loss (C 16 H 30 O, 238.23 Da, can improve the identification scale of S-palmitoylation modified peptides. According to the search results of open search, it is found that there is crosstalk between the lipid modifications of proteins (including different modified amino acids and different connection modes of lipid modifications, or different carbon chain lengths and different saturation degrees of the same lipid modification). That is, the same protein may have multiple lipid modifications with potential interaction, or the same site of the protein may have different fatty acid (such as C16:0, C16:1, etc.) modification states at the same time.

Claims

1. A method for high throughput analysis of intact S-palmitoylated modified proteins / peptides segments, characterized in that, It comprises, The application discloses a method for selectively separating and enriching S-palmitoylation modified peptides in a biological sample by using a fluorinated graphite material as a solid carrier and through twice enrichment strategies, and establishes a sensitive mass spectrometry analysis method for the S-palmitoylation modified peptides by using different acetonitrile concentration combinations to dissolve the sample and improved database searching parameters. The method comprises the following steps: (1) enriching the S-palmitoylation modified peptide sample by using the fluorinated graphite material; (2) step-by-step dissolving the modified peptide sample obtained in the step (1); 2. The method of claim 1, wherein: (3) performing chromatographic separation and mass spectrometry analysis on the S-palmitoylation modified peptides, and identifying the amino acid sequence, modification site and modification group of the S-palmitoylation modified peptides by database searching.

3. The method of claim 1, wherein In the method, the S-palmitoylation modified peptide sample is dissolved in 90% methanol aqueous solution, and then diluted with the methanol aqueous solution so that the final concentration of the peptide and the methanol is about 0.5 mg / mL and 50% respectively; the fluorinated graphite material pre-washed by methanol is added according to the amount of the peptide in the sample, and then incubated at room temperature for 2 hours; the supernatant is discarded after centrifugation, and the fluorinated graphite material is sequentially washed with 20% acetonitrile, 50% methanol and 60% methanol; the peptide is sequentially eluted from the fluorinated graphite material by using 90% methanol and 100% methanol, and the eluate is freeze-dried; the freeze-dried peptide sample is completely dissolved in the methanol solution, and the fluorinated graphite material is added to complete the second enrichment; the washing liquid is 50% methanol and 60% methanol, the eluting liquid is 90% methanol and 100% methanol, the peptide eluate is collected, and then freeze-dried for later use. In the step (2), the sample is step-by-step dissolved, which comprises the following steps: the obtained palmitoylation modified peptide sample is suspended at room temperature for 2 hours, and then dissolved in 15% ACN+0.1% FA, 35% ACN+0.1% FA and 55% ACN+0.1% FA respectively. In the step (2), the sample is step-by-step dissolved, which comprises the following steps: the obtained palmitoylation modified peptide sample is suspended at room temperature for 2 hours, and then dissolved in 15% ACN+0.1% FA, 35% ACN+0.1% FA and 55% ACN+0.1% FA respectively.

4. The method of claim 1, wherein The chromatographic separation conditions of the peptide segment and the parameter settings of database searching include: the chromatographic gradient of the sample dissolved in 15% ACN+0.1% FA is 0 min-30 min, 15%-40% B phase, 30 min-35 min, 40%-95% B phase, 35 min-45 min, 95% B phase; the chromatographic gradient of the sample dissolved in 35% ACN+0.1% FA is 0 min-30 min, 35%-65% B phase, 30 min-35 min, 65%-95% B phase, 35 min-45 min, 95% B phase; the chromatographic gradient of the sample dissolved in 55% ACN+0.1% FA is 0 min-25 min, 55%-85% B phase, 25 min-30 min, 85%-95% B phase, 30 min-35 min, 95% B phase; the mobile phase A is 100% H2O containing 0.1% FA, and the mobile phase B is 100% CAN containing 0.1% FA; the mass spectrum data obtained by using the CID fragmentation mode, when performing database searching, the Palmitoylation modification on cysteine is set as a variable modification, and the allowed daughter ion exists palmitoylloss with the molecular formula C16H30O and the mass of 238.23 Da.

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