Identification of periplaneta americana active extract CII-3 polypeptide and protein

Abstract

The invention provides the method for identifying the polypeptide and the protein in the active extract CII-3 of the periplaneta americana, the components such as the polypeptide and the protein can be analyzed through a nano-liter liquid phase and high-resolution mass spectrum tandem technology, and the identification work of corresponding macromolecular compounds is completed in combination with database matching. The invention fills the blank of CII-3 macromolecular substance research, enriches the material basis of CII-3, and provides macromolecular compound information for further development of related researches such as pharmacodynamic material basis, action target and the like of periplaneta americana series products taking CII-3 as a raw material.

Description

Technical Field

[0001] The invention belongs to the field of natural medicines, and particularly relates to an identification method for CⅡ-3 polypeptide and protein extracted from an active extract of American cockroaches. Background Art

[0002] The American cockroach (Periplaneta americana L.), commonly known as the "cockroach," also known as the "American cockroach" or "oil thief," belongs to the dried form of the genus Periplaneta, family Blattaria, order Blattaria, order Pterygota, class Insecta. An extract from this insect, CⅡ-3, exhibits anti-tumor, immunomodulatory, and anti-inflammatory activities. Its chemical components primarily include amino acids, nucleosides, peptides, and proteins. Current research on the chemical composition of CⅡ-3 has focused on the identification of small molecules, while the composition of CⅡ-3 peptides and proteins remains unclear.

[0003] The rapid development of analytical methods based on high-throughput mass spectrometry has provided new approaches and technologies for the analysis and identification of macromolecular compounds. Nanoliquid chromatography coupled with high-resolution mass spectrometry can be used to analyze components such as peptides and proteins, and combined with database matching to complete the identification of corresponding macromolecular compounds. Summary of the Invention

[0004] The main purpose of the present invention is to provide a method for identifying CⅡ-3 polypeptides and proteins in active extracts of American cockroaches, so as to fill the gap in the research of CⅡ-3 macromolecules, enrich the material basis of CⅡ-3, and provide macromolecular compound information for further research on the pharmacological material basis, target sites, etc. of American cockroach series products.

[0005] The technical solution of the present invention is as follows: A method for identifying polypeptides in an American cockroach extract CⅡ-3, comprising the following steps:

[0006] 1) Sample pretreatment: Weigh an appropriate amount of lyophilized powder of American cockroach extract CⅡ-3, add the extract, sonicate on ice, centrifuge, aspirate the supernatant, ultrafilter, collect the filtrate, and freeze-dry the eluate to a dry powder;

[0007] 2) Peptide desalting: Use Solution A to activate the desalting column, use Solution B to equilibrate the desalting column, use Solution B to dissolve the lyophilized powder obtained in step 1) pretreatment, add it to the desalting column, and allow the sample to slowly flow through the desalting column. The peptide is captured by the desalting column, and other non-hydrophobic small molecules such as salt flow out and are discarded. Then, add Solution C to clean the desalting column to wash away residual salts, add Solution A to elute the peptide, and freeze-dry the eluate;

[0008] 3) Peptide LC-MS / MS analysis;

[0009] 4) Database search;

[0010] 5) Bioinformatics analysis.

[0011] In some embodiments, the extracting solution in step 1) is a 0.1% TFA aqueous solution.

[0012] In some embodiments, the ultrasonic condition in step 1) is 100W and ultrasonication for 10s.

[0013] In some embodiments, the centrifugation condition in step 1) is 4° C., 17,000 g for 10 min.

[0014] In some embodiments, the ultrafiltration conditions in step 1) are as follows: aspirate 400 μL of supernatant using a 10 KDa ultrafiltration tube, ultrafilter to 100 μL at 10,000 g, repeatedly aspirate 400 μL of supernatant, continue ultrafiltration to 40 μL, collect the filtrate, and discard residual proteins and other macromolecules with a molecular weight greater than 10 KDa in the ultrafiltration tube.

[0015] In some embodiments, the solution a in step 2) is 200 μL of 0.1% TFA, 80% acetonitrile solution.

[0016] In some embodiments, the solution b in step 2) is a 0.1% TFA, 1% acetonitrile solution.

[0017] In some embodiments, the solution c in step 2) is a 0.1% TFA, 0.5% acetonitrile solution.

[0018] In some embodiments, the chromatographic conditions in step 3) are: using an EASY-nLC 1000 ultra-high pressure nanoliter liquid chromatography system. The chromatographic column is Acclaim PepMap C 18 The column was 75 μm × 250 mm in size; mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The sample was dissolved in 40 μL of mobile phase A, and 2 μL of sample was drawn up by the autosampler and loaded onto the analytical column at a flow rate of 500 nL / min. The sample was separated by gradient elution on the analytical column at a flow rate of 300 nL / min. The relevant liquid phase gradient is as follows:

[0019]

[0020] In some embodiments, the mass spectrometry conditions in step 3) are as follows: detection is performed using an Orbitrap Fusion mass spectrometer. The ion source is electrospray (ESI); the spray voltage is 2.4 kV; the heated capillary temperature is 320°C; the scanning mode is positive ion detection, and data-dependent detection (DDA) is used to automatically switch between MS and MS / MS acquisition. The primary mass spectrometry scan range is m / z 300-1500, with a primary mass spectrometry resolution of 120,000; the secondary mass spectrometry scan range is m / z 110-2000, with a secondary mass spectrometry resolution of 15,000. The specific secondary scan range is automatically controlled based on the mass-to-charge ratio of the parent ion, and high-energy induced dissociation (HCD) is used to fragment the parent ion.

[0021] A method for identifying proteins in an American cockroach extract CⅡ-3 comprises the following steps:

[0022] i) protein enzymatic hydrolysis;

[0023] ii) LC-MS / MS analysis;

[0024] iii) database search;

[0025] iv) bioinformatics analysis;

[0026] In some embodiments, the enzymatic hydrolysis method in step i) is: weigh an appropriate amount of freeze-dried powder of American cockroach extract CⅡ-3, add an appropriate amount of SDT lysis buffer, heat in a boiling water bath for 5 minutes, cool to room temperature, and centrifuge to obtain the supernatant; add 200μL U Abuffer to mix, transfer the solution to a 10KD ultrafiltration centrifuge tube, centrifuge at 12000g for 15 minutes, repeat twice, and discard the filtrate; add an appropriate amount of IAA to the residue, shake at 600rpm for 1 minute, place at room temperature in the dark for 30 minutes, centrifuge at 12000g for 10 minutes, add 100μL U UA buffer, centrifuge at 12000g for 10 minutes, repeat twice, add 100μL NH4HCO3 buffer, centrifuge at 14000g for 10 minutes, repeat twice, add 40μL Trypsin buffer to the residue, shake at 600rpm for 1 minute, and place at 37°C for 16-18 hours. Replace the collection tube, centrifuge at 12000g for 10min, and collect the filtrate; the peptide fragments after enzymatic hydrolysis are separated by C 18 The stage tip was desalted and vacuum dried. After drying, the peptides were reconstituted with 0.1% FA and the peptide concentration was determined by OD280 for LC-MS / MS analysis.

[0027] Preferably, the SDT lysis buffer is composed of 4% SDS, 100mM DTT, 100mM Tris-HCl; the UA buffer is composed of 8M Urea, 150mM Tris-HCl, pH 8.0; the IAA is composed of 50mM IAA in UA; and the Trypsin buffer is composed of 6μg Trypsin in 40μL NH4HCO3 buffer.

[0028] In some embodiments, the chromatographic conditions in step ii) are as follows: an EASY-nLC 1200 ultrahigh pressure nanoliter liquid chromatography system is used. The chromatographic column is an EASY-Spray column, 75 μm × 150 mm, 3 μm; mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a 0.1% formic acid-80% acetonitrile-water mixture; the sample is separated by gradient elution on the analytical column at a flow rate of 300 nL / min. The relevant liquid phase gradient is as follows:

[0029]

[0030] In some embodiments, the mass spectrometry conditions in step 3) are as follows: detection is performed using a Q-Exactive HF-X mass spectrometer. The ion source is electrospray (ESI); the scanning mode is positive ion detection, and analysis is performed in data-dependent mode (DDA). The primary mass spectrometer scan range is m / z 350-1800, and the primary mass spectrometer resolution is 60,000. The secondary mass spectrometer analysis is acquired according to the following method: the 20 most intense ions in the primary mass spectrum are selected and the parent ion is fragmented by high-energy induced dissociation (HCD), and the secondary mass spectrometer resolution is 15,000.

[0031] In the present invention, appropriate amount means that the amount of each component is within the range of reasonable experiment according to the experimental purpose.

[0032] The beneficial effects of the present invention are as follows: the present invention provides a method for identifying polypeptides and proteins in the active extract CⅡ-3 of American cockroaches, fills the gap in the research of CⅡ-3 macromolecules, enriches the material basis of CⅡ-3, and provides macromolecular compound information for further research on the pharmacological material basis, action targets, etc. of American cockroach series products using CⅡ-3 as raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Peptide identification flow chart

[0034] Figure 2 Total ion current chromatogram of the identification of American cockroach CⅡ-3 peptide in positive ion mode of electrospray ionization source

[0035] Figure 3Secondary mass spectrum and structural analysis of peptide DGIAELIKLESSL

[0036] Figure 4 Secondary mass spectrum and structural analysis of peptide EIHSIIGLPPFVPP

[0037] Figure 5 Secondary mass spectrum and structural analysis of peptide FGSTLLDVIQSGLENH

[0038] Figure 6 Secondary mass spectrum and structural analysis of peptide AIYLGDILRGLNLNPTQA

[0039] Figure 7 Secondary mass spectrum and structural analysis of the peptide DLTDYLMKILTER

[0040] Figure 8 GO functional annotation enrichment analysis of CⅡ-3 peptides belonging to precursor proteins

[0041] Figure 9 Subcellular localization statistics of CⅡ-3 peptide precursor protein

[0042] Figure 10 COG functional annotation statistics of CⅡ-3 peptides belonging to precursor proteins

[0043] Figure 11 Protein identification flow chart

[0044] Figure 12 Base peak chromatogram of the American cockroach CⅡ-3 protein identification in the positive ion mode of electrospray ionization source

[0045] Figure 13 Secondary mass spectrum and structural analysis of the corresponding peptide fragment of QBH70326.1 protein

[0046] Figure 14 Secondary mass spectrum and structural analysis of the corresponding peptide fragment of AAC34312.1 protein

[0047] Figure 15 Secondary mass spectrum and structural analysis of the corresponding peptide fragment of AVR99436.1 protein

[0048] Figure 16 Secondary mass spectrum and structural analysis of the corresponding peptide fragment of AVA17392.1 protein

[0049] Figure 17 Secondary mass spectrum and structural analysis of the corresponding peptide fragment of AAB84283.1 protein

[0050] Figure 18 Bubble diagram of GO functional annotation enrichment analysis of CⅡ-3 protein components

[0051] Figure 19 Subcellular localization statistics of CⅡ-3 protein components DETAILED DESCRIPTION

[0052] The following examples may enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the examples.

[0053] Example 1. Identification of polypeptides in the American cockroach extract CⅡ-3

[0054] The identification process of CⅡ-3 peptide from American cockroach extract is shown in Figure 1 . The following steps are included:

[0055] 1) Sample preparation

[0056] The American cockroach extract CⅡ-3 (provided by Professor Liu Guangming of Dali University) was rotary evaporated and freeze-dried to obtain a powder, which was stored at -20°C.

[0057] 2) Sample pretreatment

[0058] Weigh 5 mg of CⅡ-3 lyophilized powder and add 1.5 mL of the extract. Sonicate on ice for 10 minutes. Sonicate under the following conditions: pulse on for 5 seconds, pulse off for 15 seconds, power 100W, 4°C, and centrifuge at 17,000 g for 10 minutes. Collect 1 mL of the supernatant. Aspirate 400 μL of the supernatant and ultrafilter at 10,000 g using a 10 kDa ultrafiltration tube to a volume of 100 μL. Repeat this process by aspirating 400 μL of the supernatant and continuing the ultrafiltration process until the volume is reduced to 40 μL. Collect the filtrate and discard any residual proteins (larger molecules with a molecular weight greater than 10 kDa) in the ultrafiltration tube. Freeze-dry the eluate to a dry powder.

[0059] 3) Peptide desalting

[0060] The desalting column was activated with 200 μL of 0.1% TFA, 80% acetonitrile;

[0061] The desalting column was equilibrated with 600 μL of 0.1% TFA, 1% acetonitrile solution;

[0062] Dissolve the freeze-dried sample in 400 μL of 0.1% TFA, 1% acetonitrile solution and add it to the desalting column. Allow the sample to flow slowly through the desalting column. The peptide is captured by the desalting column, while other non-hydrophobic small molecules such as salt flow out and are discarded.

[0063] Then add 200 μL of 0.1% TFA, 0.5% acetonitrile solution to clean the desalting column and remove residual salts;

[0064] Add 300 μL of 0.1% TFA, 80% acetonitrile solution and allow the liquid to slowly flow through the desalting column to elute the polypeptide. Use a new tube to collect the eluate, and freeze-dry the eluate.

[0065] 4) LC-MS / MS analysis of peptides

[0066] The samples were separated using an EASY-nLC 1000 ultra-high pressure nanoliter liquid chromatography system. The chromatographic column was Acclaim PepMap C 18 Column (75 μm × 250 mm). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. The sample was dissolved in 40 μL of liquid, and 2 μL of sample was drawn up by the autosampler and loaded onto the analytical column at a flow rate of 500 nL / min. The sample was separated on the analytical column by gradient elution at a flow rate of 300 nL / min. The relevant liquid phase gradient is shown in Table 1.

[0067] Table 1 Liquid phase gradient

[0068] Time(min) Liquid B ratio 0 5％ 105 30％ 110 90％ 112 90％ 113 5％ 120 stop

[0069] The ion source was electrospray ionization (ESI); the spray voltage was 2.4 kV; the heated capillary temperature was 320°C; the scan mode was positive ion detection; and data-dependent acquisition (DDA) was used to automatically switch between MS and MS / MS acquisition. Full-scan MS was performed using an Orbitrap primary scan with a scan range of m / z 300–1500 and a resolution of 120,000 (at m / z 200). The maximum ion introduction time was 50 ms, and the automatic gain control (AGC) was set to 1.0 × 10 6 The precursor ions meeting the MS / MS fragmentation criteria were then fragmented using high-energy induced dissociation (HCD) within 3 seconds and scanned using an orbitrap at a resolution of 15,000. The scan range was automatically controlled based on the precursor ion mass-to-charge ratio, with a fixed minimum scan range of m / z = 110 and a maximum scan range of 2,000. The minimum ion intensity for MS / MS was set to 50,000.

[0070] The maximum ion introduction time for MS / MS was 80 ms, and the AGC control was set to 1.0×10 5 The precursor ion selection window was set to 1.6 Da. MS / MS acquisition was performed for ions with charge numbers of 2, 3, and 4, with dynamic exclusion set to perform one MS / MS run per precursor ion within 10 seconds, followed by a 21-second exclusion period and 30% collision energy.

[0071] 5) ESI mass spectrometry data

[0072] The total ion current chromatogram of the American cockroach CⅡ-3 peptide identification in the positive ion mode of the electrospray ion source is shown in the figure below. Figure 2 shown.

[0073] 6) Database search

[0074] Peaks database search: Mass spectrometry raw files (raw files) were directly searched using PEAKS Online (X Build, version 1.4.2020-10-02_113407). Parameter settings are shown in Table 2.

[0075] Table 2. Peaks analysis parameter settings

[0076]

[0077]

[0078] Peptide identification results: The Uniprot database was used for analysis, species selection: Blattidae (American cockroach family), website: https: / / www.uniprot.org / taxonomy / 6974. Protein entries: 1161 (number of annotated proteins: 151, number of unannotated proteins: 1010), download time: 2021-07-03. A total of 571 peptides were identified, which can be attributed to 329 precursor proteins. The information of the top 15 peptides with the highest mass spectrometry response intensity is shown in Table 3, the information of the top 15 precursor proteins with the highest coverage is shown in Table 4, and the secondary mass spectra of some peptides are shown in Table 3. Figure 3-7 shown.

[0079] Table 3 Detailed information of CⅡ-3 peptide

[0080]

[0081] Table 4 Information of the top 15 high coverage precursor proteins

[0082]

[0083]

[0084] 7) Bioinformatics Analysis

[0085] Peptide Gene Ontology (GO) analysis: The precursor protein was analyzed by GO using the Uniprot database, and GO annotation enrichment analysis was performed from three aspects: biological process (BP), molecular function (MF), and cellular component (CC). The results are shown in Figure 8According to the top 10 GO annotations, the molecular functions mainly involve protein heterogeneity, protein dimerization, and arginine kinase activity; the cellular components mainly involve the cytoplasmic matrix, extracellular region, and G protein-coupled receptor heterodimer complex; and the biological processes mainly involve negative regulation of vesicle fusion, negative regulation of synaptic vesicle fusion to the presynaptic active zone membrane, and positive regulation of heart rate.

[0086] Subcellular localization analysis: Results are shown in Figure 9 The largest number of precursor proteins were located in the cell membrane (51 proteins, accounting for about 40.48%), followed by the cytoplasm (29 proteins, accounting for about 23.02%) and the extracellular matrix (19 proteins, accounting for about 15.08%).

[0087] Clusters of Orthologous Groups (COG) analysis: A total of 87 precursor proteins (accounting for about 26.44%) were successfully annotated and can be divided into 13 different functional categories. Figure 10 Among them, the functions with the largest number of annotations are energy production and conversion (21, accounting for about 24.14%), signal transduction mechanism (17, accounting for about 19.54%), and cytoskeleton (11, accounting for about 12.64%).

[0088] Example 2. Identification of CⅡ-3 Protein from American Blattella Extract

[0089] The protein identification process in the American cockroach extract CⅡ-3 is shown in Figure 11 . The following steps are included:

[0090] 1) Protein enzymatic hydrolysis

[0091] Weigh an appropriate amount of lyophilized powder of American cockroach extract CⅡ-3, add an appropriate amount of SDT lysis buffer (4% SDS, 100mM DTT, 100mM Tris-HCl), heat in a boiling water bath for 5min, cool to room temperature, and centrifuge to obtain the supernatant;

[0092] The supernatant was added with 200 μL U Abuffer (8 M Urea, 150 mM Tris-HCl, pH 8.0) and mixed thoroughly. The solution was transferred to a 10KD ultrafiltration centrifuge tube and centrifuged at 12,000 g for 15 min. This was repeated twice and the filtrate was discarded.

[0093] An appropriate amount of IAA (50 mM IAA in UA) was added to the residue, shaken at 600 rpm for 1 min, placed in the dark at room temperature for 30 min, centrifuged at 12000 g for 10 min, and the filtrate was discarded;

[0094] The residue was added with 100 μL of Abuffer and centrifuged at 12,000 g for 10 min, repeated twice, and the filtrate was discarded;

[0095] The residue was added with 100 μL NH 4 HCO 3 buffer and centrifuged at 14 000 g for 10 min, repeated twice, and the filtrate was discarded;

[0096] Add 40 μL of trypsin buffer (6 μg trypsin in 40 μL NH₄HCO₃ buffer) to the residue, shake at 600 rpm for 1 min, and incubate at 37°C for 16-18 h. Replace the collection tube, centrifuge at 12,000 g for 10 min, and collect the filtrate.

[0097] The peptide fragments after enzymatic digestion were used 18 The stage tip was desalted and vacuum dried. After drying, the peptides were reconstituted with 0.1% FA and the peptide concentration was determined by OD280 for LC-MS / MS analysis.

[0098] 2) LC-MS / MS analysis

[0099] Sample separation was performed using an EASY-nLC 1200 ultrahigh-pressure nanoliter liquid chromatography system. The chromatographic column was an EASY-Spray column (75 μm × 150 mm, 3 μm). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of a mixture of 0.1% formic acid and 80% acetonitrile in water. Samples were separated on the analytical column by gradient elution at a flow rate of 300 nL / min. The relevant liquid phase gradient is shown in Table 5.

[0100] Table 5 Liquid phase gradient

[0101] Time(min) Liquid B ratio 0 2％ 2 5％ 44 28％ 51 40％ 53 100％ 69 100％ 69 stop

[0102] After sample separation, a Q-Exactive HF-X mass spectrometer was used for data-dependent acquisition (DDA). The ion source was electrospray ionization (ESI); the scanning mode was positive ion detection; the primary mass spectrometer scan range was m / z 350–1800, the primary mass spectrometer resolution was set to 60,000 (at m / z 200), and the automatic gain control (AGC) was set to 3×10 6 Secondary mass spectrometry was performed as follows: the 20 most intense ions were selected from the primary mass spectrometer and the parent ions were fragmented by high energy induced dissociation (HCD). The secondary mass spectrometry resolution was 15000. The AGC was set to 1×10 5 , the maximum ion introduction time for MS / MS was 50 ms, the precursor ion selection window was set to 1.6 Da; the collision energy was 28.

[0103] 3) ESI mass spectrometry data

[0104] The base peak chromatogram of the American cockroach CⅡ-3 protein identification in the positive ion mode of the electrospray ion source is shown in the figure below. Figure 12 shown.

[0105] 4) Database search

[0106] The mass spectrometry database search software used was MaxQuant 1.6.17.0; the following protein database was used: ncbi-Periplaneta americana (porgn_txid 6978)-1419-20220714.fasta, obtained from https: / / www.ncbi.nlm.nih.gov / protein, with protein entry 1419, downloaded on July 14, 2022. The MaxQuant database search software analysis parameters are shown in Table 6.

[0107] Table 6 MaxQuant analysis parameter settings

[0108]

[0109] Protein identification results: After mass spectrometry data retrieval, PSM FDR≤0.01 and Protein FDR≤0.01 were used as the screening criteria for peptide, site, and protein identification, respectively. A total of 418 peptides were obtained, corresponding to 83 known proteins (the top 15 high-coverage protein information is shown in Table 7). Table 8 shows the information of the top 15 peptides with the highest mass spectrometry response intensity. The secondary mass spectra of the corresponding peptides of some proteins are shown in Figure 8. Figure 13-17 shown.

[0110] Table 7 Information of the top 15 proteins with high coverage

[0111]

[0112] Table 8 Detailed information on peptide identification in protein detection (first 15 entries)

[0113]

[0114] 5) Bioinformatics Analysis

[0115] Protein GO analysis: GO annotation analysis was performed on the 83 identified proteins, and annotations were made from three aspects: cellular component (CC), molecular function (MF) and biological process (BP). Figure 18 The top 10 entries with significant enrichment in the three branches of biological processes, cellular components, and molecular functions are displayed. Biological processes mainly include organic matter metabolism, primary metabolism, and organic nitrogen compound metabolism; cellular components mainly include protein-containing complexes, intracellular anatomical structures, and cytoplasm; and molecular functions mainly include ion binding, hydrolase activity, and calcium ion binding.

[0116] Subcellular localization analysis: Figure 19 It can be seen that the subcellular localization of most proteins is in the cell membrane (10, 62.5%), and the remaining proteins are located in the cytoplasm, ribosomes, and extracellular regions.

Claims

1. A method for identifying polypeptides in an American cockroach extract CⅡ-3, comprising the following steps: 1) Sample pretreatment: Weigh an appropriate amount of lyophilized powder of American cockroach extract CⅡ-3, add the extract, sonicate on ice, centrifuge, aspirate the supernatant, ultrafilter, collect the filtrate, and freeze-dry the eluate to a dry powder; 2) Peptide desalting: Use Solution A to activate the desalting column, use Solution B to equilibrate the desalting column, use Solution B to dissolve the lyophilized powder obtained in step 1) pretreatment, add it to the desalting column, and allow the sample to slowly flow through the desalting column. The peptide is captured by the desalting column, and other non-hydrophobic small molecules such as salt flow out and are discarded. Then, add Solution C to clean the desalting column to wash away residual salts, add Solution A to elute the peptide, and freeze-dry the eluate; 3) Peptide LC-MS / MS analysis; 4) Database search; 5) Bioinformatics analysis.

2. The polypeptide identification method according to claim 1, characterized in that The extract in step 1) is a 0.1% TFA aqueous solution.

3. The polypeptide identification method according to claim 1, characterized in that The ultrasonic condition in step 1) is 100W and 10s; preferably, the centrifugation condition in step 1) is 4°C and 17000g for 10min.

4. The polypeptide identification method according to claim 1, characterized in that The ultrafiltration conditions in step 1) are as follows: 400 μL of supernatant is drawn using a 10 KDa ultrafiltration tube, ultrafiltered to 100 μL at 10,000 g, 400 μL of supernatant is repeatedly drawn, ultrafiltration is continued to 40 μL, the filtrate is collected, and residual proteins and other macromolecules with a molecular weight greater than 10 KDa in the ultrafiltration tube are discarded.

5. The polypeptide identification method according to claim 1, characterized in that In step 2), solution a is 200 μL of 0.1% TFA, 80% acetonitrile solution; solution b in step 2) is 0.1% TFA, 1% acetonitrile solution; and solution c in step 2) is 0.1% TFA, 0.5% acetonitrile solution.

6. The polypeptide identification method according to claim 1, characterized in that The chromatographic conditions in step 3) are as follows: using an EASY-nLC 1000 ultra-high pressure nanoliter liquid chromatography system; the chromatographic column is Acclaim PepMap C 18 Column, size 75 μm × 250 mm; mobile phase A is 0.1% formic acid in water, mobile phase B is 0.1% formic acid in acetonitrile; The sample was dissolved in 40 μL of mobile phase A. After 2 μL of sample was aspirated by the autosampler, it was loaded onto the analytical column at a flow rate of 500 nL / min. The sample was separated by gradient elution on the analytical column at a flow rate of 300 nL / min. The relevant liquid phase gradient is as follows:

7. The polypeptide identification method according to claim 1, characterized in that The mass spectrometry conditions in step 3) are as follows: detection using an Orbitrap Fusion mass spectrometer; the ion source is electrospray ESI; the spray voltage is 2.4 kV; the heated capillary temperature is 320° C.; the scanning mode is positive ion detection, and the data-dependent mode DDA is used to automatically switch between MS and MS / MS acquisition; the primary mass spectrometry scanning range is m / z 300-1500, and the primary mass spectrometry resolution is 120,000; the secondary mass spectrometry scanning range is m / z 110-2000, and the secondary mass spectrometry resolution is 15,000. The specific range of the secondary scan is automatically controlled according to the mass-to-charge ratio of the parent ion, and the parent ion is fragmented using high-energy induced dissociation (HCD).

8. A method for identifying proteins in an American cockroach extract CⅡ-3, comprising the following steps: i) protein enzymatic hydrolysis; ii) LC-MS / MS analysis; iii) database search; iv) bioinformatics analysis; The enzymatic hydrolysis method in step i) is as follows: weigh an appropriate amount of lyophilized powder of American cockroach extract CⅡ-3, add an appropriate amount of SDT lysis solution, heat in a boiling water bath for 5 minutes, cool to room temperature, and centrifuge to obtain the supernatant; add 200 μL of U Abuffer and mix well, transfer the solution to a 10KD ultrafiltration centrifuge tube, centrifuge at 12000g for 15 minutes, repeat twice, and discard the filtrate; add an appropriate amount of IAA to the residue, shake at 600 rpm for 1 minute, stand at room temperature in the dark for 30 minutes, centrifuge at 12000g for 10 minutes, add 100 μL of U Abuffer, centrifuge at 12000g for 10 minutes, repeat twice, add 100 μL of NH4HCO3 buffer, centrifuge at 14000g for 10 minutes, repeat twice, add 40 μL of Trypsin buffer to the residue, shake at 600 rpm for 1 minute, and stand at 37°C for 16-18 hours; replace the collection tube, centrifuge at 12000g for 10 minutes, and collect the filtrate; the peptide fragments after enzymatic hydrolysis are separated by filtration using C 18 The Stage Tip is desalted and vacuum dried; after drying, the peptides are re-dissolved with 0.1% FA, and the peptide concentration is determined by OD280 for LC-MS / MS analysis; preferably, the SDT lysis buffer is composed of 4% SDS, 100mM DTT, 100mM Tris-HCl; the UA buffer is composed of 8M Urea, 150mM Tris-HCl, pH 8.0; the IAA is composed of 50mM IAAin UA; and the Trypsin buffer is composed of 6μg Trypsin in 40μL NH4HCO3 buffer.

9. The protein identification method according to claim 8, characterized in that The chromatographic conditions in step ii) are as follows: an EASY-Spray column, 75 μm × 150 mm, 3 μm; mobile phase A is 0.1% formic acid in water, and mobile phase B is a mixture of 0.1% formic acid and 80% acetonitrile in water; the sample is separated by gradient elution on the analytical column at a flow rate of 300 nL / min. The relevant liquid phase gradient is as follows:

10. The protein identification method according to claim 8, characterized in that The mass spectrometry conditions in step 3) are as follows: the ion source is electrospray (ESI); the scanning mode is positive ion detection, and the analysis is performed in data-dependent mode (DDA); the primary mass spectrometry scanning range is m / z 350-1800, and the primary mass spectrometry resolution is 60,000; and the secondary mass spectrometry analysis is collected according to the following method: the 20 most intense ions in the primary mass spectrum are selected and the parent ions are fragmented by high-energy induced dissociation (HCD), and the secondary mass spectrometry resolution is 15,000.

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