Edible bird's nest characteristic peptide, screening method and application thereof

Abstract

The present application relates to a kind of characteristic peptide segments that can be used for identifying the authenticity of bird's nest, which comprises 21 peptide segment amino acid sequences.In identifying the authenticity of bird's nest, the sample to be tested is subjected to enzymatic hydrolysis, followed by reduction and alkylation, and then subjected to high performance liquid chromatography separation, and the obtained protein peptides are compared with the above-mentioned characteristic peptide segments by high-resolution mass spectrometry analysis.The present application also provides a screening method for the above-mentioned characteristic peptide segments.The present application identifies 21 characteristic peptide segments from bird's nest, greatly enriching the number of characteristic peptide segments of bird's nest, and based on proteomics technology, different molecular weight bird's nest proteins and peptide segments are identified using in-gel digestion method, and a screening method for characteristic peptide segments of bird's nest is established.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a screening method for characteristic peptide segments of bird's nest based on proteomics technology and application. BACKGROUND

[0002] Edible bird's nest generally refers to the nest built by the saliva and feathers of six bird species of the genus Aerodramus in the family Apedidae during the breeding process. Meanwhile, the nests of some bird species of the genus Aerodramus in the family Apedidae can also be used as bird's nest. Bird's nest has a long history in China, and there are records of eating bird's nest as early as the Tang Dynasty. Until now, people have been eating bird's nest as a high-grade health product.

[0003] From the composition, bird's nest mainly contains proteins, carbohydrates, lipids, some inorganic elements, vitamins, hormones, etc. Some studies have analyzed the amino acids contained in bird's nest and found that it contains 20 common amino acids such as aspartic acid and glutamic acid, does not contain hydroxyproline and sarcosine, and contains 8 essential amino acids. The content of carbohydrates in bird's nest is second only to protein, mainly including sialic acid, mannose, glucosamine, galactosamine, etc. Lipids account for about 0.14-0.28% of bird's nest, and the fatty acid composition includes stearic acid, palmitic acid, linolenic acid, and linoleic acid. Bird's nest contains a high content of inorganic elements, and contains essential trace elements for humans. Studies have shown that the elements with high content in bird's nest are four common elements Ca>Mg>Na>K, followed by five essential trace elements Fe>Zn>Mn>Cr>Cu.

[0004] However, bird's nest itself is a complex system, and although it has been studied for many years, the material basis of bird's nest is still not clear, and there are still cases of counterfeiting or using inferior products. In 2011, a food safety incident of "blood bird's nest" counterfeiting led to a large amount of nitrite, which had a serious impact on the bird's nest industry and exposed the problem that the quality and safety of bird's nest could not be guaranteed to a large extent. The identification and quality control of bird's nest have also been paid more attention to. With the development of various analysis techniques and molecular biology, the research on bird's nest has also been more in-depth.

[0005] At present, researchers have developed various methods for the identification of the authenticity of bird's nest, which can qualitatively identify bird's nest by using ultraviolet light irradiation and specific chemical reactions. Various instrumental analysis techniques can also be used for the identification of bird's nest, such as infrared spectroscopy, high-performance liquid chromatography, etc. The development of molecular biology provides new techniques for the study of bird's nest. Molecular biology techniques can not only be used for the analysis of bird's nest itself, but also can identify the species source. The main techniques used for the analysis of bird's nest are real-time fluorescent PCR and two-dimensional electrophoresis.

[0006] Proteins are the main components of bird's nest, and the study of proteins in bird's nest is the focus of bird's nest research. Proteomics technology, as a new technology that has developed rapidly in recent years, provides an important reference for the study of bird's nest. The concept of proteomics was proposed in 1994, which means "all proteins expressed by the genome of a cell or a tissue". It is a tool discipline that studies how proteins function as a whole. After more than 20 years of development, proteomics has become one of the most important disciplines for studying proteins and their functions, and plays an important role in drug analysis and drug target discovery.

[0007] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is an important method for protein separation and the first step of proteomics research. It can separate proteins according to their molecular weights. The separated proteins can be cut and analyzed by subsequent mass spectrometry. If proteins are separated according to their charges in another direction, it is two-dimensional electrophoresis (2D-PAGE). Some proteomics studies have identified proteins in bird's nest. In 2010, researchers used two-dimensional electrophoresis to preliminarily analyze bird's nest. In 2012, Liu et al. reported the results of a bird's nest proteomics study based on two-dimensional electrophoresis. This was the first detailed study of bird's nest proteomics. The authors identified a protein in bird's nest, which was AMCase-like protein. However, due to the lack of protein data for the golden-crowned flying fox in the database, this protein was identified from a turkey protein database that had some homology with the golden-crowned flying fox.

[0008] In 2019, a method for identifying bird's nest-specific peptides using proteomics was reported. The method first grinds the bird's nest into powder in liquid nitrogen and uses 7M urea + 2M thiourea for protein extraction. After extraction, 120mM DTT and 600mM IAA are added to reduce and alkylate the proteins. Then, trypsin is added for proteolysis, and the proteins are digested into peptides.

[0009] The hydrolyzed peptide segments were analyzed by nanoliter liquid chromatography tandem quadrupole-time of flight mass spectrometry, and the peptide segment source was determined after comparison with protein database search. The sample of bird's nest and the species database used for different adulterants were Apodidae, Sus, Tremella fuciformis, Gallus, Cypriniformes. Subsequently, a preliminary quantitative analysis was carried out by using a multiple reaction monitoring (MRM) method. The proteomic search results showed that Lysyl oxidase 3, Acidic mammalian chitinase, Actin, cytoplasmic type 5, Hemojuvelin, Deleted in malignant brain tumors 1 protein, Actin, gamma-entericsmooth muscle and other proteins were identified in multiple different bird's nest samples. But the corresponding proteins were also identified in the adulterants of tremella, fish swim bladder, egg white and other foods.

[0010] The peptide segments consisting of 6-20 amino acids without any deletion or modification were used as screening conditions, and 72 characteristic peptide segments were found in bird's nest and adulterant species. The selected peptide segments in bird's nest were TLFTVLVK, EMVAAFEQEAR, SLWSCPYR, SSEWGTICDDR, IWLDNVNCAGGEK.

[0011] Then, the LC-QTRAP-MS system was used to verify all the candidate peptide segments, and the peptide segments TLFTVLVK and EMVAAFEQEAR had higher response values in the bird's nest peptide segments, about 10 times of the other three peptide segments. The MRM method was used to find 32 stable MRM transitions under the CID fragmentation condition, of which 10 were from egg white, 6 were from fish swim bladder, 6 were from pig skin, and 9 were from bird's nest. The quantitative characteristics of these fragment ions were evaluated, and the linear experiment showed that the 32 stable MRM transitions were potential quantifiable fragment ions. After correction, it was found that the linear optimization of the EMVAAFEQEAR peptide segment in the bird's nest was optimal.

[0012] The technology developed an identification method of bird's nest characteristic peptide segments, which can distinguish bird's nest and its adulterants, and can also carry out preliminary quantification. However, the technology has the following shortcomings:

[0013] 1. The species database for proteomic search is less. Only one species database was used for matching of bird's nest sample, which may lead to less information.

[0014] 2. The technology directly carries out in-solution protease digestion, which cannot judge the information of bird's nest proteins and peptide segments with different molecular weights.

[0015] 3. The technology only relies on one proteomics operation to find corresponding peptide segments, and the reliability is relatively low. SUMMARY

[0016] In order to overcome the above technical defects, the most important technical purpose of the present application is to provide characteristic peptide segments for identifying the authenticity of bird's nest.

[0017] The second purpose of the present application is to provide the application of the characteristic peptide segments of the bird's nest in the identification of the authenticity of the bird's nest.

[0018] The third purpose of the present application is to provide a bird's nest protein and peptide segment traceability database.

[0019] The fourth purpose of the present application is to provide a method for identifying the authenticity of the bird's nest.

[0020] The fifth purpose of the present application is to provide a screening method of the characteristic peptide segments.

[0021] The characteristic peptide segments of the bird's nest, the amino acid sequence of which is shown in any one of SEQ ID No. 1-21:

[0022]

[0023]

[0024] The application of the characteristic peptide segments of the bird's nest in the identification of the authenticity of the bird's nest.

[0025] A bird's nest protein and peptide segment traceability database, comprising one or more peptide segments shown in any one of SEQ ID No. 1-21.

[0026] Preferably, the bird's nest protein and peptide segment traceability database further contains the protein and peptide segment database of chimney swift (Chaetura pelagica), horned tree swallow (Hemiprocne comata) and house swallow (Hirundo rustica rustica).

[0027] Preferably, the edible bird's nest protein and peptide traceability database further comprises a protein and peptide database of species related to edible bird's nest and adulterants. For example, Anas platyrhynchos, Anser anser, Asarcornis scutulata, Aythya fuligula, Bos mutus, Bos taurus, Capra hircus, Carassius auratus, Carassius gibelio, Cyprinus carpio, Danio rerio, Gallus gallus, Glycine max, Labeo calbasu, Labeo rohita, Onychostoma macrolepis, Ovis aries, Sinocyclocheilus anshuiensis, Sus scrofa, Tremella fuciformis, and Tremella mesenterica. The peptides not belonging to Aerodramus maximus, Aerodramus papuensis, and Hirundo rustica in the sample can be considered as adulterants.

[0028] Preferably, the edible bird's nest protein and peptide traceability database can be established by mixing proteins from edible bird's nest raw materials from different origins, edible bird's nest products of different brands, and different adulterants, performing reduction, alkylation, and trypsin digestion, and performing qualitative proteomics research, and establishing all protein and peptide information obtained by the research.

[0029] A method for identifying the authenticity of edible bird's nest, comprising the following steps:

[0030] (1) adding a lysis solution to the protein of the sample, mixing with acetone, and collecting the precipitate; adding tetraethylammonium bromide (TEAB) to the precipitate, dispersing the precipitate, and then adding trypsin for enzymatic digestion; and then reducing with dithiothreitol (DTT) and alkylating with iodoacetamide (IAA) to obtain a protein extract;

[0031] (2) dissolving the protein extract obtained in step (1) with mobile phase A, and then performing high performance liquid chromatography, and then performing high resolution mass spectrometry analysis, wherein the high performance liquid chromatography uses mobile phase A of 0.1% formic acid (FA) and 2% acetonitrile (ACN) in water, and mobile phase B of 0.1% formic acid and 90% acetonitrile in water, and the gradient elution program of the mobile phase is as follows:

[0032] Time Mobile phase A Mobile phase B 0 - 68 min 77％～94％ 6％～23％ 68 - 82 min 68％～77％ 23％～32％ 82 - 86 min 20％～68％ 32％～80％ 86 - 90 min 20％ 80％ ;

[0033] (3) searching and analyzing the data obtained from the high resolution mass spectrometer to identify whether it contains a characteristic peptide segment as shown in any one of the amino acid sequences of SEQ ID No. 1-21.

[0034] Preferably, the trypsin in step (1) is added at a mass ratio of 1:50 to the protein.

[0035] Preferably, the method for dispersing the precipitate in step (1) is by ultrasonic dispersion.

[0036] Preferably, in the gradient elution in step (2), the flow rate of the mobile phase is 500 nL / min.

[0037] Preferably, the ion source of the high resolution mass spectrometer in step (2) is NSI ion source; the mass analyzer is Orbitrap Exploris TM 480 mass spectrometer.

[0038] Preferably, the high resolution mass spectrometer in step (2) is a two-stage tandem mass spectrometer.

[0039] Preferably, in the two-stage tandem mass spectrometer, the data acquisition mode uses a data-dependent scanning (DDA) program; that is, after the first-stage scanning, the top 25 peptide segment parent ions with the highest signal intensity are selected to enter the HCD collision cell in turn, and a fragmentation energy of 27% is used for fragmentation, and the same is sequentially subjected to secondary mass spectrometry.

[0040] Preferably, in the two-stage tandem mass spectrometer, the first-stage mass spectrometry scanning range is set to 400-1200 m / z, and the scanning resolution is set to 60000.

[0041] Preferably, in the two-stage tandem mass spectrometer, the fixed starting point of the secondary mass spectrometry scanning range is 110 m / z, the secondary scanning resolution is set to 15000, and TurboTMT is set to Off.

[0042] Preferably, the automatic gain control (AGC) is set to 100%, the signal threshold is set to 5E4 ions / s, the maximum injection time is set to Auto, and the dynamic exclusion time of tandem mass spectrometry scanning is set to 20 s to avoid repeated scanning of the parent ion.

[0043] Preferably, the secondary mass spectrometry data is searched by Proteome Discoverer.

[0044] Preferably, the searching analysis in step (3) is performed in the database of the edible bird's nest protein and peptide fragments.

[0045] The application further provides a screening method of the characteristic peptide fragments, comprising the following steps:

[0046] (1) Constructing the database of the edible bird's nest protein: mixing the edible bird's nest raw materials from different origins, the edible bird's nest products of different brands and the proteins of different adulterants together, performing reduction, alkylation and trypsin digestion, performing qualitative proteomics research, and establishing all the protein and peptide fragment information obtained by the research as the database of the edible bird's nest protein;

[0047] (2) Screening the characteristic peptide fragments of the edible bird's nest: performing proteomics research on several real edible bird's nest samples, after polyacrylamide gel electrophoresis, cutting the gel, reduction, alkylation and in-gel digestion, obtaining the edible bird's nest proteins of different molecular weights, and screening the 21 peptide fragments after comparison and analysis with the database of step (1).

[0048] The screening method, wherein the method of the qualitative proteomics research in step (1) specifically comprises:

[0049] (a) equal amount of protein of each sample is subjected to enzymolysis, the volume is adjusted to be consistent with the lysis solution, 1 volume of pre-cooled acetone is added, vortexed and mixed, then 4 volumes of pre-cooled acetone is added, precipitated at 20℃ for 2h, centrifuged at 4500g for 5min, the supernatant is discarded, the precipitate is washed with pre-cooled acetone for 2 times, the precipitate is dried, then TEAB with a final concentration of 200mM is added, the precipitate is dispersed by ultrasonic, trypsin is added at a ratio of 1:50 (protease: protein, m / m), and enzymolysis is performed overnight; dithiothreitol (DTT) is added to make the final concentration 5mM, and reduction is performed at 56℃ for 30min; then iodacetamide (IAA) is added to make the final concentration 11mM, and incubation is performed at room temperature in the dark for 15min;

[0050] (b) the obtained peptide fragments are dissolved with mobile phase A, and then separated by using an EASYnLC1200 ultra-high performance liquid system; mobile phase A is a water solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is a water solution containing 0.1% formic acid and 90% acetonitrile; the liquid phase gradient is set as 6%-23% B for 0-68min, 23%-32% B for 68-82min, 32%-80% B for 82-86min, 80% B for 86-90min, and the flow rate is maintained at 500nL / min; after the peptide fragments are separated by the ultra-high performance liquid system, ionization is performed in the NSI ion source, and then the OrbitrapExploris TM480Mass spectrometry was used for analysis; ion source voltage was set to 2.3 kV, FAIMS compensation voltage was set to 2.3 kV; the first mass spectrometry scan range was set to 400-1200 m / z, and the scan resolution was set to 60000; the second mass spectrometry scan range was set to 110 m / z, the second scan resolution was set to 15000, and TurboTMT was set to Off; the data acquisition mode used a data-dependent scan (DDA) program, i.e., after the first scan, the top 25 peptide precursor ions with the highest signal intensity were selected to enter the HCD collision cell in turn, and were fragmented using 27% fragmentation energy, and were subjected to secondary mass spectrometry analysis in turn; the automatic gain control (AGC) was set to 100%, the signal threshold was set to 5E4 ions / s, the maximum injection time was set to Auto, the tandem mass spectrometry scan dynamic exclusion time was set to 20 s to avoid repeated scanning of the parent ions; and the secondary mass spectrometry data was searched using Proteome Discoverer (v2.4.1.15).

[0051] Preferably, the search parameters of the secondary mass spectrometry data in step (b) are set as follows: a decoy database is added to the database to calculate the false positive rate (FDR) caused by random matching, and a common contaminant database is added to the database to eliminate the influence of contaminant proteins in the identification results; the enzyme digestion mode is set to Trypsin (Full); the number of missed cleavage sites is set to 2; the minimum length of the peptide is set to 6 amino acid residues; the maximum number of peptide modifications is set to 3; the mass error tolerance of the primary parent ion is set to 10 ppm, and the mass error tolerance of the secondary fragment ion is set to 0.02 Da; Carbamidomethyl (C) is set as a fixed modification, and Oxidation (M), Acetyl (N-terminus), Met-loss (M), and Met-Met-loss+acetyl (M) are set as variable modifications; the FDRs of protein, peptide, and PSM identification are all set to 1%.

[0052] The screening method described above, wherein the method of proteomic research in step (2) specifically comprises:

[0053] (A) 5xloading buffer was added to different bird's nest protein solutions, and the solutions were treated in a 95℃ metal bath for 5-10 min; the cooked samples were added to SDS-PAGE for gel electrophoresis, and the voltage was 80 V for 0-15 min and 120 V for 15-70 min; the gel was dyed with coomassie brilliant blue dye and decolorized with a decolorizing solution;

[0054] (B) Wash the decolorized and extracted gel with high purity water twice, determine the part of the gel that needs to be cut according to the picture, prepare a 1.5 ml centrifuge tube; rinse the centrifuge tube with ethanol, remove the liquid at the bottom of the tube after standing; rinse the glass plate, knife and a 100 μl gun head with ethanol, stand until dry; change gloves, place the gel on the glass plate, cut the required part and cut into 1-1.5 mm square small particles, and put them into the corresponding tube;

[0055] (C) Add 500 μl of decolorizing agent, vortex slightly, and decolorize at low speed; centrifuge and remove the liquid when the gel particles are completely colorless and transparent; add 500 μl of dehydrating agent, vortex slightly, and dehydrate at low speed for 10 min or in the refrigerator; centrifuge and remove the liquid when the gel particles are completely white; add 50 μl of 10 mM DTT, incubate at 56°C for 30-40 min; centrifuge and remove the liquid; add 50 μl of 55 mM IAA, incubate at room temperature in the dark for 60 min; centrifuge and remove the liquid; wash the gel particles with 500 μl of high purity water, vortex, and centrifuge to remove the liquid; add 500 μl of dehydrating agent, vortex slightly, and dehydrate at low speed for 10 min; add enough trypsin according to 1 μl per 1 mm of gel particles, and put it in the refrigerator for 45-60 min for complete swelling; add 50 mM ammonium bicarbonate (ABC) according to 2 μl per 1 mm of gel strip, and incubate at 30°C overnight for 12-16 h;

[0056] (D) Add 50 μl of extraction solution: 50% ACN 5% FA, shake at high speed at room temperature for 25 min, centrifuge and transfer the liquid to a new labeled 1.5 ml centrifuge tube; repeat the extraction once, combine the extracts, and concentrate under vacuum;

[0057] (E) Before loading, dissolve the sample with liquid chromatography mobile phase A, centrifuge at 15000 rpm for 45 min, and take the supernatant to the sample bottle; use an EASYnLC1200 ultra-high performance liquid system for separation; mobile phase A is 0.1% formic acid in water; mobile phase B is 0.1% formic acid in acetonitrile; the liquid phase gradient is set as follows: 0-2 min 4%-12% B; 2-23 min 12%-30% B; 23-27 min 30%-50% B; 27-28 min 50%-100% B, the flow rate is maintained at 300 nL / min; after the peptide segment is separated by the ultra-high performance liquid system, it is injected into the NSI ion source for ionization, and then enters the Orbitrap Fusion Lumos mass spectrometer for analysis; the ion source voltage is set to 2.2 kV, and the peptide segment parent ion and its secondary fragments are detected and analyzed using high-resolution Orbitrap; the primary mass spectrometry scan range is set to 375-1500 m / z, and the scan resolution is set to 120000; the secondary mass spectrometry scan range is fixed to 100 m / z, the secondary scan resolution is set to 30000; and the secondary mass spectrometry data is searched using pFind3.

[0058] Preferably, in the step (C), the decoloring agent is 50% ACN 25-50mM ABC; the dehydrating agent is ACN; and the sufficient trypsin is 1-10ng / ul 50mM ABC.

[0059] Preferably, in the primary mass spectrum, the data acquisition mode uses a data-dependent scan (DDA) program, that is, after the primary scan, the top 25 peptide segment parent ions with the highest signal intensity are selected into the HCD collision cell in turn, and are fragmented using a fragmentation energy of 30%, and the secondary mass spectrum analysis is performed in turn; the automatic gain control (AGC) is set to 100%, the signal threshold is set to 5E4 ions / s, the maximum injection time is set to 100ms, and the dynamic exclusion time of the tandem mass spectrum scan is set to 30s to avoid repeated scanning of the parent ions.

[0060] Preferably, the search parameters of the secondary mass spectrum are set as follows: the database is Chaetura pelagica+Hemiprocne comata+Hirundo rustica rustica, the reverse library is added to calculate the false positive rate (FDR) caused by random matching, and the common contamination library is added to the database to eliminate the influence of contaminant proteins in the identification results; the enzyme cutting mode is set to Trypsin (Full); the number of missed cutting sites is set to 3; the minimum length of the peptide segment is set to 6 amino acid residues; the maximum number of peptide segment modifications is set to 3; the mass error tolerance of the primary parent ion is set to 20ppm, the mass error tolerance of the secondary fragment ion is set to 0.02Da; Carbamidomethyl (C) is set as a fixed modification, Oxidation (M), Acetyl (AnyN-term), Met-loss (ProteinN-termM),

[0061] MetMet-loss+Acetyl (ProteinN-termM) are set as variable modifications; the FDR of protein, peptide segment and PSM identification are all set to 1%.

[0062] The present application adopts the above-mentioned screening method, and after repeated screening, 42 proteins and 130 peptide segments are identified in total after the 01-08 samples are combined and searched. In the proteins, the 12 proteins with PSM≥10 are selected according to the number of peptide segments (PSM) matched to the protein, and the maximum PSM is 62. Among them, there are proteins with similar or identical names, and the reason is that their species are different.

[0063] Then all the peptide segments matched to these proteins were listed. Further, according to the sequence of the peptide segments, the repetitions were removed by regarding the same sequence as the same peptide segment and only keeping the data with the smallest protein number. There were 68 different peptide segments in the 12 proteins.

[0064] In order to further reduce the number of candidate peptide segments, the 68 peptide segments were compared with the peptide segments identified in the true and false base library data, and 28 peptide segments were found which were also identified in the base library. Among them, 21 peptide segments were identified three times. The 21 peptide segments can finally be used as the signature peptide segments of bird's nest, which are from different bird's nest proteins.

[0065] The technical effects that can be achieved by the present application by adopting the above technical solutions include:

[0066] 1. 21 characteristic peptide segments are identified from bird's nest, which greatly enriches the number of characteristic peptide segments of bird's nest. The defects of the prior art that the species database for proteomics search is less and the information is less are overcome.

[0067] 2. Based on the proteomics technology, different molecular weight bird's nest proteins and peptide segments are identified by in-gel digestion method, and a screening method for bird's nest characteristic peptide segments is established. The technology can judge the information of different molecular weight bird's nest proteins and peptide segments, and has high reliability. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 The flow chart for establishing the bird's nest protein traceability database;

[0069] Figure 2 The number of proteins (1167) and the number of peptide segments (5435) identified in the database establishment;

[0070] Figure 3 The number of proteins identified in different species;

[0071] Figure 4 The SDS-PAGE map of bird's nest protein;

[0072] Figure 5 The number of bird's nest proteins identified in in-gel digestion samples;

[0073] Figure 6 The 21 bird's nest characteristic peptide segments screened based on the proteomics method. DETAILED DESCRIPTION

[0074] To further illustrate the present application, the following examples are provided. Unless otherwise specified, the reagents used in the examples below are commercially available and the databases used in the examples below are publicly available online databases. The following examples are illustrative only and are not intended to limit the present application.

[0075] Example 1: Establishment of edible bird's nest traceability protein database

[0076] In this example, an edible bird's nest protein traceability database was first constructed. Edible bird's nest raw materials from different origins, edible bird's nest products of different brands, and proteins of different adulterants were mixed together, reduced, alkylated, and trypsin-cleaved, and qualitative proteomic studies were performed. Figure 1 Flow chart for the establishment of the edible bird's nest protein traceability database.

[0077] Research methods:

[0078] An equal amount of protein from each sample was subjected to enzymolysis, and the volume was adjusted to be consistent with the lysis solution. One volume of pre-cooled acetone was added, vortexed to mix, and then 4 volumes of pre-cooled acetone were added. The precipitate was allowed to settle at 20°C for 2 hours. Centrifugation was performed at 4500g for 5 minutes, and the supernatant was discarded. The precipitate was washed twice with pre-cooled acetone. After the precipitate was air-dried, tetraethylammonium bromide (TEAB) was added to a final concentration of 200 mM. The precipitate was dispersed by ultrasonic, and trypsin was added at a ratio of 1:50 (protease: protein, m / m) for overnight enzymolysis. Dithiothreitol (DTT) was added to a final concentration of 5 mM, and the mixture was reduced at 56°C for 30 minutes. Then, iodoacetamide (IAA) was added to a final concentration of 11 mM, and the mixture was incubated at room temperature in the dark for 15 minutes.

[0079] The peptides were dissolved in mobile phase A and separated using an EASYnLC1200 ultra-high performance liquid system. Mobile phase A was a solution of 0.1% formic acid and 2% acetonitrile in water; mobile phase B was a solution of 0.1% formic acid and 90% acetonitrile in water. The liquid phase gradient was set as follows: 0-68 min 6%~23% B; 68-82 min 23%~32% B; 82-86 min 32%~80% B; 86-90 min 80% B, with a flow rate of 500 nL / min. After separation by the ultra-high performance liquid system, the peptides were injected into the NSI ion source for ionization, and then entered the Orbitrap Exploris TM480Mass spectrometry was performed. The ion source voltage was set to 2.3 kV, the FAIMS compensation voltage was set to -45 V and -65 V, and both the peptide fragment parent ions and their secondary fragments were detected and analyzed using high-resolution Orbitrap. The primary mass spectrometry scan range was set to 400-1200 m / z, and the scan resolution was set to 60000; the secondary mass spectrometry scan range was set to 110 m / z, the secondary scan resolution was set to 15000, and the TurboTMT was set to Off. The data acquisition mode used a data-dependent scan (DDA) program, i.e., after the primary scan, the top 25 peptide fragment parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn, and were fragmented using 27% fragmentation energy, and secondary mass spectrometry analysis was performed in turn. In order to improve the effective utilization rate of mass spectrometry, the automatic gain control (AGC) was set to 100%, the signal threshold was set to 5E4 ions / s, the maximum injection time was set to Auto, and the dynamic exclusion time of tandem mass spectrometry scan was set to 20 s to avoid repeated scanning of parent ions.

[0080] The secondary mass spectrometry data was searched using Proteome Discoverer (v2.4.1.15). The search parameters were set as follows: the database was all the species databases shown in Table 1, the reverse database was added to calculate the false positive rate caused by random matching (FDR), and common contamination databases were added to the database to eliminate the influence of contaminant proteins in the identification results; the enzyme cutting mode was set to Trypsin (Full); the number of missed cleavage sites was set to 2; the minimum length of the peptide was set to 6 amino acid residues; the maximum number of peptide modifications was set to 3; the mass error tolerance of the primary parent ion was set to 10 ppm, and the mass error tolerance of the secondary fragment ion was set to 0.02 Da. Carbamidomethyl (C) was set as a fixed modification, and Oxidation (M), Acetyl (N-terminus), Met-loss (M), and Met-Met-loss+acetyl (M) were set as variable modifications. The FDRs of protein, peptide, and PSM identification were all set to 1%.

[0081] In order to improve the protein scale contained in the database, during the protein database search, the present scheme selected databases of multiple related species for integration (Table 1), which included all possible species related to bird's nest and adulterants. The species related to bird's nest included chimney swift (Chaetura pelagica), horned tree swift (Hemiprocne comata), and house swift (Hirundo rustica rustica).

[0082]

[0083] Using this database, 1167 proteins and 5435 peptides were identified in 3 replicates (as shown in Table 1), and the most proteins were identified in soybean among the corresponding species. In addition, corresponding proteins of Apus nipalensis, Apus pacificus and Apus nipalbipes were also identified (as shown in Table 2). Figure 2 Figure 3

[0084] This example established a database of edible-nest bird's nest proteins and peptide segments, which contains all proteins and peptide segments that can be introduced during the production of edible-nest bird's nest, and gives their species attribution. This database can be used to identify whether the edible-nest bird's nest is adulterated, for example, if peptide segments that do not belong to Apus nipalensis, Apus pacificus and Apus nipalbipes are detected in the sample, it can be considered that the edible-nest bird's nest sample is adulterated.

[0085] Example 2: Edible-nest bird's nest protein identification based on in-gel digestion-proteomics

[0086] In order to identify the characteristics of edible-nest bird's nest proteins of different molecular weights and screen for marker peptide segments of edible-nest bird's nest, in-gel digestion-proteomics identification was performed in this example. First, polyacrylamide gel electrophoresis (SDS-PAGE) was performed on different edible-nest bird's nest raw materials (Y1-Y8) and finished products (C1, J1, J3). From the electrophoretogram, it can be seen that the edible-nest bird's nest proteins of different molecular weights are distributed, and there are larger bands in 100-150 kDa in the raw materials and finished products (as shown in Table 3). Figure 4

[0087] Thereafter, different bands (bands 1-8) were subjected to gel cutting, reduction, alkylation and in-gel digestion, and proteomics analysis was performed. In the database search process, the databases of Apus nipalensis, Apus pacificus and Apus nipalbipes were simultaneously searched, which was consistent with the previous scheme. The results showed that 8, 9 and 23 different proteins were identified in the databases of the three different species, respectively (as shown in Table 4). Figure 5 Different numbers of proteins were identified in different bands, and 9, 18, 11, 24, 11, 7, 15 and 12 proteins were identified in bands 1-8, respectively, as well as 23, 31, 33, 32, 15, 11, 32 and 18 peptide segments (Table 2). The proteins identified in different bands were compared with their molecular weights (Table 3), and CHIA chitinase, Acidic mammalian chitinase, Lysyl oxiase homolog, MUC5AC and other proteins were found at different positions.

[0088] Research methods:

[0089] Edible-nest bird's nest protein solutions corresponding to the raw materials (Y1-Y8) and finished products (C1, J1, J3) were obtained by protein extraction, respectively. The extraction method is as follows: ​​​

[0090] The sample was first ground into dry powder in liquid nitrogen, 0.15-0.2 g of dry powder was weighed, 5 ml of 8M urea was added and mixed, 1 ml was ultrasonically broken, centrifuged at 15000 g for 30 min, and the supernatant was taken. The supernatant was precipitated with 5 times the amount of cold acetonitrile, centrifuged at 800 rpm for 10 min after 4 h, the supernatant was discarded, the precipitate was washed once with cold acetonitrile, and was resuspended with 8M urea or 50mM ammonium bicarbonate (ABC), and then the protein concentration was determined with a BCA protein quantitative kit.

[0091] Loading buffer (5x) was added to different bird's nest protein solutions, and they were treated in a 95°C metal bath for 5-10 min. The cooked samples were added to SDS-PAGE for gel electrophoresis. Voltage: 80V for 0-15 min; 120V for 15-70 min. The gel was stained with coomassie brilliant blue staining solution and decolorized with decolorizing solution.

[0092] The decolorized and extracted gel was washed twice with high-purity water, the part to be cut was determined according to the picture, and a 1.5 ml centrifuge tube was prepared. The centrifuge tube was rinsed with ethanol, and the liquid at the bottom of the tube was removed after standing. The glass plate, knife blade, and a 100 μl gun tip were rinsed with ethanol, and stood until dry. New gloves were put on, the gel was placed on the glass plate, the desired part was cut off, and was cut into 1-1.5 mm square small particles and put into the corresponding tube.

[0093] Add 500 μl of decolorizing agent (50% ACN 25-50 mM ABC) and slightly vortex, and shake at low speed to decolorize. When the gel particles are completely colorless and transparent, centrifuge and remove the liquid. Add 500 μl of dehydrating agent (ACN) and slightly vortex, shake at low speed for 10 min, or place in the refrigerator to dehydrate. When the gel particles are completely white, centrifuge and remove the liquid. Add 10 mM 50 μl DTT (or 1 mm tape 10 μl in proportion), and incubate at 56°C for 30-40 min. Centrifuge to remove the liquid. Add 55 mM 50 μl IAA (or 1 mm tape 10 μl in proportion), and incubate at room temperature in the dark for 60 min. Centrifuge to remove the liquid, wash the gel particles with 500 μl of high-purity water, vortex, and centrifuge to remove the liquid. Add 500 μl of dehydrating agent, slightly vortex, and shake at low speed for 10 min. When the gel particles are white, centrifuge and remove the liquid. Add enough trypsin (1-10 ng / μl 50 mM ABC) per 1 mm gel particle, and place in the refrigerator for 45-60 min to swell completely. Add 50 mM ABC at a ratio of 2 μl per 1 mm gel strip, and incubate at 30°C overnight for 12-16 h.

[0094] Add 50 μl of extraction solution (50% ACN 5% FA), shake at high speed at room temperature for 25 min, centrifuge and transfer the liquid to a new labeled 1.5 ml centrifuge tube. Repeat the extraction once, combine the extracts, and concentrate under vacuum.

[0095] Before loading, the sample was dissolved with liquid chromatography mobile phase A and centrifuged at 15000 rpm for 45 min, and the supernatant was taken to the sample bottle. Separation was performed using an EASYnLC1200 ultra-high performance liquid system. Mobile phase A was a solution of 0.1% formic acid in water; mobile phase B was a solution of 0.1% formic acid in acetonitrile. The liquid phase gradient was set as follows: 0-2 min 4%~12% B; 2-23 min 12%~30% B; 23-27 min 30%~50% B; 27-28 min 50%~100% B, and the flow rate was maintained at 300 nL / min. After the peptide fragments were separated by the ultra-high performance liquid system, they were injected into the NSI ion source for ionization, and then entered the Orbitrap Fusion Lumos mass spectrometer for analysis. The ion source voltage was set to 2.2 kV, and both the peptide fragment parent ions and their secondary fragments were detected and analyzed using high-resolution Orbitrap. The primary mass spectrometry scan range was set to 375-1500 m / z, and the scan resolution was set to 120000; the secondary mass spectrometry scan range was fixed to 100 m / z, and the secondary scan resolution was set to 30000. The data acquisition mode used a data-dependent scanning (DDA) program, that is, after the primary scan, the top 25 peptide fragment parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn, and were fragmented using 30% fragmentation energy, and secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization rate of mass spectrometry, the automatic gain control (AGC) was set to 100%, the signal threshold was set to 5E4 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of tandem mass spectrometry scan was set to 30 s to avoid repeated scanning of the parent ions.

[0096] The secondary mass spectrometry data was searched using pFind3. The search parameters were set as follows: the database was Chaetura pelagica+Hemiprocne comata+Hirundo rustica rustica, the reverse library was added to calculate the false positive rate caused by random matching (FDR), and the common pollution library was added to the database to eliminate the influence of pollution proteins in the identification results; the enzyme digestion mode was set to Trypsin (Full); the number of missed cleavage sites was set to 3; the minimum length of the peptide was set to 6 amino acid residues; the maximum number of peptide modifications was set to 3; the mass error tolerance of the primary parent ion was set to 20 ppm, and the mass error tolerance of the secondary fragment ion was set to 0.02 Da. Carbamidomethyl (C) was set as a fixed modification, and Oxidation (M), Acetyl (AnyN-term), Met-loss (ProteinN-termM),

[0097] MetMet-loss + Acetyl (Protein N-term M) is set as variable modification. FDR of protein, peptide, PSM identification is set as 1%.

[0098]

[0099]

[0100] Example 3: Screening of Edible Bird's Nest Characteristic Peptide

[0101] In this example, screening of Edible Bird's Nest characteristic peptide was carried out. In order to select the signature peptide that can be used for identification, the 01-08 samples were combined and searched in the database. The database source and search parameters were the same as in Example 1. A total of 42 proteins and 130 peptides were identified. Among the proteins, the number of peptides (PSM) matched to the protein was sorted, and 12 proteins with PSM≥10 were selected, of which the maximum PSM was 62. Among them, there were proteins with similar or identical names due to different species.

[0102]

[0103] All peptides matched to these proteins were listed. Further, according to the peptide sequence, duplicates were removed. As long as the sequence was the same, it was considered to be the same peptide, and only the data with the smallest protein number were retained. Among the 12 proteins, 68 different peptides were obtained.

[0104] In order to further reduce the number of candidate peptides, the 68 peptides were compared with the peptides identified in the true and false database in 3.1.1 (Base database), and 28 peptides were found to be also identified in the Base database, of which 21 peptides were identified three times. These 21 peptides can be used as signature peptides of Edible Bird's Nest (as shown in Table 2), which are from different Edible Bird's Nest proteins. Figure 6

[0105] The present application identified 21 characteristic peptides from Edible Bird's Nest, which greatly enriched the number of characteristic peptides of Edible Bird's Nest. Overcame the defects of less species database and less information in proteomics search in the prior art. The screening method of the present application is based on proteomics technology, which identifies Edible Bird's Nest proteins and peptides of different molecular weights by in-gel digestion method, and establishes a screening method for characteristic peptides of Edible Bird's Nest. This technology can determine the information of Edible Bird's Nest proteins and peptides of different molecular weights, and has high reliability.

[0106] ​While the application has been described and illustrated in detail, it should be understood that the foregoing description is in no way intended to be limiting. Numerous variations, substitutions, and equivalents will now be apparent to those skilled in the art without departing from the spirit and scope of the application as defined in the following claims. Furthermore, the scope of the application is not intended to be limited to the particular examples described in the specification. The application as described and claimed herein is widely applicable to processes, devices, means, methods, and steps that perform substantially the same function or achieve substantially the same result as those described in the specification. Accordingly, the appended claims are intended to include within their scope all processes, devices, means, methods, and steps that do not depart from the spirit and scope of the application.

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] SEQUENCE LISTING <110> Beijing Xiaoxian Duan Biological Technology Co., Ltd. <120> Characteristic peptide segment of yunao, screening method and application thereof <141> 2022-05-24 <160> 21 <170> SIPOSequenceListing 1.0 <210> 1 <211> 11 <212> PRT <213> Characteristic sequence <400> 1 Glu Met Val Ala Ala Phe Glu Gin Glu Ala Arg 1 5 10 <210> 2 <211> 11 <212> PRT <213> FEATURE <400> 2 Phe Ser Thr Met Val Ser Thr Pro Gin Asn Arg 1 5 10 <210> 3 <211> 11 <212> PRT <213> FEATURE <400> 3 Lys Leu Leu Val Gly Phe Pro Thr Tyr Gly Arg 1 5 10 <210> 4 <211> 10 <212> PRT <213> FEATURE <400> 4 Leu Leu Val Gly Phe Pro Thr Tyr Gly Arg 1 5 10 <210> 5 <211> 14 <212> PRT <213> FEATURE <400> 5 Thr Leu Leu Ala lie Gly Gly Trp Asn Phe Gly Thr Ala Lys 1 5 10 <210> 6 <211> 11 <212> PRT <213> FEATURE <400> 6 Glu Gly Lys Tyr Pro Leu lie Thr Thr Leu Lys 1 5 10 <210> 7 <211> 8 <212> PRT <213> FEATURE <400> 7 Tyr Pro Leu lie Thr Thr Leu Lys 1 5 <210> 8 <211> 11 <212> PRT <213> Characterizing Sequence <400> 8 Gly Asn Glu Trp Val Gly Tyr Asp Asn lie Lys 1 5 10 <210> 9 <211> 10 <212> PRT <213> Characterizing Sequence <400> 9 Phe Ser Ser Gin lie His Asn Asn Gly Arg 1 5 10 <210> 10 <211> 9 <212> PRT <213> Characterizing Sequence <400> 10 Leu Ala Asn Trp Pro Tyr Gly His Arg 1 5 <210> 11 <211> 16 <212> PRT <213> Characterizing Sequence <400> 11 Asp Ser Asn Val lie Glu Thr Glu Gin Ser His Val Glu Glu Val Arg 1 5 10 15 <210> 12 <211> 13 <212> PRT <213> Characterizing Sequence <400> 12 Arg Gin Leu Pro Val Thr Glu Gly lie Val Glu Val Arg 1 5 10 <210> 13 <211> 21 <212> PRT <213> Characterizing Sequence <400> 13 Cys Ala Gly His Glu Met Ser Leu Asn His Cys Gin His His Gly Thr 1 5 10 15 Ser Leu Asn Cys Arg 20 <210> 14 <211> 10 <212> PRT <213> FEATURE <400> 14 Cys Tyr Gin Gly Cys He Thr Thr Ser Arg 1 5 10 <210> 15 <211> 11 <212> PRT <213> FEATURE <400> 15 Phe Val Arg Lys Ser Pro Ser Val Tyr Leu Lys 1 5 10 <210> 16 <211> 8 <212> PRT <213> FEATURE <400> 16 Lys Ser Pro Ser Val Tyr Leu Lys 1 5 <210> 17 <211> 11 <212> PRT <213> FEATURE <400> 17 Cys Gly Leu Thr Asp Thr Glu Thr Cys Leu Lys 1 5 10 <210> 18 <211> 11 <212> PRT <213> FEATURE <400> 18 Gly Leu Glu Gly Cys Tyr Pro His Cys Pro Lys 1 5 10 <210> 19 <211> 10 <212> PRT <213> Characterizing Sequence <400> 19 Leu Glu Gly Ala Val lie Glu Leu Thr Arg 1 5 10 <210> 20 <211> 13 <212> PRT <213> Characterizing Sequence <400> 20 Gln Cys Ala His Ala Gly Gly Gln Pro Leu Asn Trp Arg 1 5 10 <210> 21 <211> 12 <212> PRT <213> Characterizing Sequence <400> 21 Ser Leu Ser Glu Pro Asp Val Thr Cys Ser Thr Lys 1 5 10

Claims

1. A method for authenticating bird's nest, characterized in that, Includes the following steps: (1) Add lysis buffer to the protein sample to be tested, add acetone and mix, collect the precipitate; add tetraethylammonium bromide to the precipitate to disperse the precipitate, add trypsin for enzymatic hydrolysis; then reduce with dithiothreitol and alkylate with iodoacetamide to obtain protein extract; (2) The protein extract obtained in step (1) is dissolved in mobile phase A and then subjected to high performance liquid chromatography (HPLC) followed by high resolution mass spectrometry (HPLC). In the HPLC, mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile, and mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The gradient elution program for the mobile phase is as follows: ； (3) Search and analyze the data obtained by high-resolution mass spectrometry to identify whether it contains characteristic peptides as shown in the amino acid sequences of SEQ ID No. 1 to 21; The screening method for characteristic peptides in bird's nest includes the following steps: (1) Construct a traceability database for bird's nest protein: Mix bird's nest raw materials from different origins, bird's nest finished products from different brands, and proteins from different adulterants together, perform reduction, alkylation, and trypsin digestion, conduct qualitative proteomics research, and establish a traceability database for bird's nest protein based on all the protein and peptide information obtained from the research. (2) Screening characteristic peptides of bird's nest: Proteomics studies were conducted on several real bird's nest samples. After polyacrylamide gel electrophoresis, bird's nest proteins of different molecular weights were obtained by gel cutting, reduction, alkylation and in-gel enzymatic digestion. After comparison and screening with the database analysis in step (1), characteristic peptides with amino acid sequences as shown in SEQ ID No. 1 to 21 were obtained. The qualitative proteomics research methods in step (1) of the screening method for characteristic peptides in bird's nest include: (a) Take equal amounts of protein samples for enzymatic digestion, and adjust the volume to be consistent with the lysis buffer; add 1 volume of pre-cooled acetone, vortex to mix, and then add 4 volumes of pre-cooled acetone, and precipitate at 20℃ for 2 h; centrifuge at 4500g for 5 min, discard the supernatant, and wash the precipitate twice with pre-cooled acetone; after drying the precipitate, add tetraethylammonium bromide to a final concentration of 200 mM, sonicate to disperse the precipitate, add trypsin at a protein-to-protein mass ratio of 1:50, and digest overnight; add dithiothreitol to a final concentration of 5 mM, and reduce at 56℃ for 30 min; then add iodoacetamide to a final concentration of 11 mM, and incubate at room temperature in the dark for 15 min to obtain the peptide to be tested. (b) The peptide obtained in step (a) was dissolved in mobile phase A of liquid chromatography and then separated using an EASYnLC1200 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient settings were: 0-68 min 6%–23% mobile phase B; 68-82 min 23%–32% mobile phase B; 82-86 min 32%–80% mobile phase B; 86-90 min 80% mobile phase B, with the flow rate maintained at 500 nL / min. After separation by the ultra-high performance liquid chromatography system, the peptide was injected into an NSI ion source for ionization and then introduced into the Orbitrap Exploris ionization system. TM 480 m / z mass spectrometry was used for analysis; the ion source voltage was set to 2.3 kV FAIMS compensation voltage; the primary mass spectrometry scan range was set to 400-1200 m / z, and the scan resolution was set to 60000; the secondary mass spectrometry scan range had a fixed starting point of 110 m / z, and the secondary scan resolution was set to 15000, with TurboTMT set to Off; a data-dependent scanning program was used for data acquisition, i.e., after the primary scan, the top 25 peptide precursor ions with the highest signal intensity were selected and sequentially entered into the HCD collision cell, fragmented using 27% fragmentation energy, and then analyzed by secondary mass spectrometry in the same manner; automatic gain control was set to 100%, the signal threshold was set to 5E4 ions / s, the maximum injection time was set to Auto, and the dynamic exclusion time for tandem mass spectrometry scans was set to 20 s to avoid repeated scanning of precursor ions; secondary mass spectrometry data were searched using ProteomeDiscoverer (v2.4.1.15); The proteomics research method in step (2) of the screening method for characteristic peptides of bird's nest includes: (A) Add 5× loading buffer to different bird's nest protein solutions and treat in a 95℃ metal bath for 5-10 min; add the cooked samples to SDS-PAGE for gel electrophoresis, voltage: 80V for 0-15 min; 120V for 15-70 min; stain the gel with Coomassie brilliant blue staining solution and destain with destaining solution; (B) Wash the decolorized and image-extracted gel twice with high-purity water. Determine the part to be cut according to the image and prepare a 1.5ml centrifuge tube. Rinse the centrifuge tube with ethanol, let it stand, and remove the liquid at the bottom of the tube. Rinse the glass plate, blade, and a 100μl pipette tip with ethanol and let it stand to dry. Put on new gloves, place the gel on the glass plate, cut off the required part, and cut it into small particles of 1-1.5mm square and put them into the corresponding tubes. (C) Add 500 μl of decolorizing agent and vortex briefly, then shake at low speed to decolorize; once all the gel particles are colorless and transparent, centrifuge and remove the liquid; add 500 μl of dehydrating agent and vortex briefly, then shake at low speed for 10 min or place in a refrigerator to dehydrate; once all the gel particles are white, centrifuge and remove the liquid; add 10 mM 50 μl of dithiothreitol and incubate at 56 °C for 30-40 min; centrifuge to remove the liquid; add 55 mM Incubate with 50 μl of iodoacetamide in the dark at room temperature for 60 min; centrifuge to remove liquid, wash the gel particles with 500 μl of high-purity water, vortex, centrifuge to remove liquid; add 500 μl of dehydrating agent, vortex briefly, shake at low speed for 10 min, until the gel particles turn white, centrifuge and remove liquid; add sufficient trypsin at a rate of 1 μl per 1 mm gel particle, refrigerate for 45-60 min to fully swell, add 50 mM ammonium bicarbonate at a ratio of 2 μl per 1 mm gel strip, incubate overnight at 30℃ for 12-16 h; (D) Add 50 μl of extraction buffer: 50% acetonitrile and 5% formic acid. Shake at high speed for 25 min at room temperature, centrifuge, and transfer the liquid to a newly labeled 1.5 ml centrifuge tube. Repeat the extraction once, combine the extracts, and concentrate by vacuum centrifugation. (E) Before loading the sample, dissolve the sample in liquid chromatography mobile phase A and centrifuge at 15000 rpm for 45 min. Collect the supernatant into a sample vial. Separate the sample using an EASYnLC1200 ultra-high performance liquid chromatography system. Mobile phase A is an aqueous solution containing 0.1% formic acid; mobile phase B is acetonitrile containing 0.1% formic acid. Liquid phase gradient settings: 0-2 min 4%–12% B; 2-23 min 12%–30% B; 23-27 min 30%–50% B; 27-28 min 50%–100% B, with the flow rate maintained at 300 nL / min. After separation by the ultra-high performance liquid chromatography system, the peptide fragments are injected into an NSI ion source for ionization and then introduced into an Orbitrap Fusion system. Lumos mass spectrometry was used for analysis; the ion source voltage was set to 2.2 kV, and the peptide precursor ion and its secondary fragments were detected and analyzed using high-resolution Orbitrap; the primary mass spectrometry scan range was set to 375-1500 m / z, and the scan resolution was set to 120,000; the secondary mass spectrometry scan range had a fixed starting point of 100 m / z, and the secondary scan resolution was set to 30,000; the secondary mass spectrometry data were retrieved using pFind3.

2. The method for authenticating bird's nest according to claim 1, characterized in that: The trypsin mentioned in step (1) is added at a mass ratio of 1:50 to the protein; The ion source for the high-resolution mass spectrometry mentioned in step (2) is an NSI ion source; The mass analyzer is an orbital ion trap analyzer; The high-resolution mass spectrometry mentioned in step (2) is a two-stage tandem mass spectrometry.

3. The method for authenticating bird's nest according to claim 2, characterized in that: In the second-stage tandem mass spectrometry, a data-dependent scanning procedure is used for data acquisition. The mass spectrometry scanning range was set to 400-1200 m / z, and the scanning resolution was set to 60000. The starting point of the secondary mass spectrometry scanning range is fixed at 110 m / z, and the secondary scanning resolution is set to 15000.

4. The method for identifying the authenticity of bird's nest according to claim 1, characterized in that, In step (b), the retrieval parameters for the secondary mass spectrometry data are set as follows: a reverse library is added to the database to calculate the false positive rate caused by random matching, and a common contamination library is added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme digestion method is set to Trypsin (Full); the number of missed cleavage sites is set to 2; the minimum peptide length is set to 6 amino acid residues; the maximum number of peptide modifications is set to 3; the mass error tolerance of the primary precursor ion is set to 10 ppm, and the mass error tolerance of the secondary fragment ion is 0.02 Da; Carbamidomethyl (C) is set as a fixed modification, and Oxidation (M), Acetyl (N-terminus), Met-loss (M), and MetMet-loss+acetyl (M) are set as variable modifications; the FDR for protein, peptide, and PSM identification is set to 1%.

5. The method for authenticating bird's nest according to claim 1, characterized in that, In step (C), the decolorizing agent is 50% acetonitrile and 25-50mM ammonium bicarbonate; the dehydrating agent is acetonitrile; and sufficient trypsin is 1-10 ng / μl of 50mM ammonium bicarbonate.

6. The method for identifying the authenticity of bird's nest according to claim 1, characterized in that, In the primary mass spectrometry, the data acquisition mode uses a data-dependent scanning procedure, that is, after the primary scan, the top 25 peptide precursor ions with the highest signal intensity are selected and sequentially introduced into the HCD collision cell, and fragmentation is performed using 30% of the fragmentation energy. Similarly, secondary mass spectrometry analysis is performed sequentially. The automatic gain control is set to 100%, the signal threshold is set to 5E4 ions / s, the maximum injection time is set to 100ms, and the dynamic exclusion time for tandem mass spectrometry scanning is set to 30s to avoid repeated scanning of precursor ions.

7. The method for identifying the authenticity of bird's nest according to claim 1, characterized in that, The secondary mass spectrometry (PSS) search parameters were set as follows: the database consisted of *Chaetura pelagica*, *Hemiprocne comata*, and *Hirundorustica rustica*. A reverse database was added to calculate the false positive rate caused by random matching, and a common contamination database was added to eliminate the influence of contaminating proteins in the identification results. The enzyme digestion method was set to Trypsin (Full); the number of missed cleavage sites was set to 3; the minimum peptide length was set to 6 amino acid residues; the maximum number of peptide modifications was set to 3; the mass error tolerance for the primary precursor ion was set to 20 ppm, and the mass error tolerance for the secondary fragment ion was 0.02 Da. Carbamidomethyl (C) was set as a fixed modification, while Oxidation (M), Acetyl (Any N-term), Met-loss (Protein N-term M), and MetMet-loss+Acetyl (Protein N-term M) were set as variable modifications. The FDR for protein, peptide, and PSM identification was set to 1%.

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