CN117517526A - Method for identifying meat adulterated components by using high performance liquid chromatography mixed labeling small peptide sample adding method - Google Patents

Abstract

The invention relates to a method for identifying meat adulterated components by utilizing a high performance liquid chromatography mixed labeling small peptide sample adding method, which is characterized in that protein structures of different animal sources are analyzed, various small peptides, oligopeptides and polypeptide fragments are produced by the protein through a proper enzymolysis cleavage mode, and characteristic peptide fragments are screened to be used as markers for identifying the meat of the animal sources. The detection method is simple and easy to operate, adopts the bioinformatics technology and combines the high-resolution mass spectrometry technology to screen the characteristic peptide fragments, establishes an LC-MS/MS method with accurate quantification, wide applicability and high flux, identifies different sources of meat sources, and provides technical support for meat authenticity and mixing detection.

Description

Method for identifying meat adulterated components by using high performance liquid chromatography mixed labeling small peptide sample adding method

Technical Field

The invention belongs to the field of food detection, and particularly relates to a method for identifying meat adulterated components by utilizing a high performance liquid chromatography mixed labeling small peptide sample adding method.

Background

On the one hand, the meat food is counterfeited and doped; on the other hand, the meat food detection technology has defects. Quantitative detection techniques suffer from serious shortcomings and it is difficult to identify whether is deliberately incorporated or unintentionally contaminated. And for deeply processed meat products with severe degradation of DNA, the PCR method is not applicable.

The meat food is rich in protein, and the primary structure information (amino acid composition sequence) of the protein has good stability and is not influenced by deep processing; the proteins from different sources of meat have unique genetic differences, peptide segment regions where the differences are located form characteristic peptide segments, and the meat source containing the proteins can be qualitatively identified and quantitatively determined by mass spectrometry technology. Providing a good solution to these problems. The peptides of different proteins differ significantly in amino acid sequence, i.e., primary structure, and these differences can be identified by high resolution mass spectrometry. In theory, the protein biological information difference is combined with mass spectrum technology, so that the detection technical problem can be solved.

The small peptide is a product containing 2-10 amino acids prepared by enzymolysis or fermentation, and has the functions of supplementing animal body amino acids, regulating appetite, improving growth performance, enhancing organism immunity, etc. The meat food is rich in protein, and the primary structure information (amino acid composition sequence) of the protein has good stability and is not influenced by deep processing; the proteins from different sources of meat have unique genetic differences, peptide segment regions where the differences are located form characteristic peptide segments, and qualitative identification and quantitative determination can be carried out on the meat sources containing the proteins by mass spectrometry technology, so that a good solution is provided for the problems. The peptides of different proteins differ significantly in amino acid sequence, i.e., primary structure, and these differences can be identified by high resolution mass spectrometry. In theory, the protein biological information difference is combined with mass spectrum technology, so that the detection technical problem can be solved.

Disclosure of Invention

The invention aims to overcome the defects of the background technology, and carry out extensive and intensive research, and the GC-MS detection method of the method for identifying the adulterated ingredients of the meat by utilizing the mixed labeling small peptide sample adding method is used for identifying the sources of the meat by adopting the bioinformatics technology and the high-resolution mass spectrometry technology based on the respective specific structures of the proteins of the meat of different sources under the condition of the defects of the meat counterfeit and adulteration detection technology, so that the invention is completed by establishing the LC-MS/MS method with accurate quantification, wide applicability and high flux for identifying the sources of the meat of different sources and detecting the authenticity and adulteration of the meat.

The method is realized by the following steps:

1) Extracting protein in meat and measuring the protein content in the extracting solution;

2) Disulfide bond reduction;

3) Sulfhydryl alkylation;

4) Removing unreacted IAA;

5) Diluting;

6) Performing enzymolysis;

7) Purifying and desalting by using an HLB small column.

Further, in the step 1), protein extraction in meat is carried out through extraction, homogenization, vortex or/and centrifugation, and supernatant fluid is treated through a filter membrane to obtain a liquid to be tested.

Further, in the step 2), disulfide bond reduction reaction occurs by vortex and vibration.

Further, in the step 6), the diluted extracting solution in the step 5) is subjected to enzymolysis, and the peptide fragment solution after enzymolysis is obtained through shaking enzymolysis.

Further, in the step 7), desalting is carried out by acetonitrile cleaning, balancing, eluting, nitrogen blowing, constant volume taking supernatant, and filtering membrane treatment is carried out to obtain the liquid to be tested to be analyzed.

In more detail, by collecting meat product samples from different sources (porcine, bovine, equine, chicken, etc.); database searching, analyzing the primary structure biological information of the target protein; the proper breaking mode, the macromolecular protein is broken into oligopeptide or polypeptide fragments by enzyme or other physical and chemical means, and the generation rule of peptide fragments is researched; analyzing the peptide fragment information, and analyzing the primary structure information of the peptide fragment by combining a time-of-flight mass spectrometer; screening the labeled peptide fragments, and screening out conservative peptide fragments with sensitive and stable signals, wherein the conservative peptide fragments are used as markers for identifying protein sources; obtaining peptide biological information through a time-of-flight mass spectrum peptide sequencing technology, comparing and matching the peptide biological information with a protein sequence database, and analyzing different source protein characteristics to mark peptide fragments; LC-MS/MS qualitative and quantitative method based on simultaneous monitoring of various exogenous proteins and hydrolysates of the identified peptide fragments is established.

The invention analyzes the protein structure of different animal sources, and makes the protein produce various small peptides, oligopeptides and polypeptide fragments by proper enzymolysis and cleavage modes, and screens characteristic peptide fragments as markers for identifying meat of the animal sources. And analyzing characteristic peptide fragments of different animal-derived proteins by adopting a high-resolution mass spectrometry technology (time-of-flight mass spectrometry), and researching basic information such as molecular weight, charge number and the like of the characteristic peptide and a fragmentation mode of the characteristic peptide. According to the information, an analysis method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) is further established, and various technologies (such as an enzyme-linked immunosorbent assay technology, a PCR technology and the like) are applied to carry out comparison verification on the method of the project research, so that the qualitative accuracy of the method is confirmed; and (5) inspecting the accuracy of the quantification of the method by a standard reference substance labeling and recycling mode.

Drawings

FIG. 1 is a TIC diagram of pork;

FIG. 2 is a diagram of beef TIC;

FIG. 3 is a TIC diagram of horse meat;

FIG. 4 is a chicken TIC diagram;

FIG. 5 is a MRM chromatogram of pork peptide fragments;

FIG. 6 is a MRM chromatogram of a beef peptide fragment;

FIG. 7 is a MRM chromatogram of chicken peptide fragments;

FIG. 8 is an MRM chromatogram of a martin fragment;

FIG. 9 is a MRM total ion flow diagram of a pork and beef mix sample;

fig. 10 is a mass spectrum of pork SALAHAVQSSR;

FIG. 11 is a mass spectrum of beef HPSDFGADAQAAMSK;

fig. 12 is a mass spectrum of pork LVVITAGAR;

FIG. 13 is a mass spectrum of beef LVIITAGAR;

fig. 14 is a mass spectrum of beef ALEDQLSELK;

FIG. 15 is a mass spectrum of YDIILLR;

fig. 16 is a mass spectrum of beef TLALLFSGPASGEAEGGPK;

fig. 17 is a mass spectrum of pork TLAFLFAER;

FIG. 18 is a mass spectrum of beef EASGPINFTVFLNMFGEK;

FIG. 19 is a 5% pork MRM chromatogram;

FIG. 20 is a 10% pork MRM chromatogram;

FIG. 21 is a 20% pork MRM chromatogram;

FIG. 22 is a 40% pork MRM chromatogram;

FIG. 23 is a 60% pork MRM chromatogram;

FIG. 24 is an 80% pork MRM chromatogram;

FIG. 25 is a graph of SALAHAVQSSR peptide standard;

FIG. 26 is a graph of TLAFLFAER peptide standard;

FIG. 27 is a standard curve of YDIILNLR peptide fragment;

FIG. 28 is a graph of LVVITAGAR peptide standard.

Detailed Description

The invention is further described below in connection with specific embodiments in order to provide a better understanding of the present technical solution.

Sample pretreatment:

1) Protein extraction in meat: accurately weighing 2g minced meat sample, adding 5mL of extraction solution (50 mM Tris-HCl,7M urea, 2M thiourea, pH 8.0), homogenizing (8000rpm 30s;9000rpm 30s;11000rpm 30s in sequence), cleaning cutter head with 5mL of extraction solution, mixing the solutions, and centrifuging at 20000rpm for 30min at 4deg.C under 10 min;

2) The approximate protein content of the extracted solution was measured and is shown in Table 1;

TABLE 1 protein content

Name of the name	Protein (g/ml)	Mg/ml
			Pig	0.0208	20.8
Cattle	0.0145	14.5
			Sheep (sheep)	0.0156	15.6
Chicken (chicken)	0.0205	20.5
			Duck	0.0167	16.7
Horse	0.0181	18.1

3) Disulfide bond reduction: taking 990 mu L of supernatant, adding 10 mu L of DTT solution (0.5M, 100mM ammonium bicarbonate as solvent), and performing vortex (the concentration of DTT in the mixed solution is 5 mM), and performing oscillation reaction for 1h at 56 ℃; the reaction temperature cannot be higher than 60 ℃, so that the carbamylation of lysine and the N-terminal of protein is avoided;

4) Thiol alkylation: after standing at room temperature, 10 μl of IAA solution (1M, 100mM ammonium bicarbonate as solvent) was added, and the mixture was vortexed and mixed (IAA concentration in the solution was 10 mM) and reacted in the dark for 30min; IAA concentration 2 times of DTT is added, IAA is decomposed by visible light, and decomposed products can have adverse effects on protein;

5) Unreacted IAA was removed: taken out, 10. Mu.L (final concentration 5 mM) of DTT solution (500 mM) was added and reacted in the dark for 15min;

6) Dilution (5-fold dilution): the dilution was 25mM Tris-HCl, pH 8.0. 200. Mu.L of extract+790. Mu.L of diluent+10. Mu.L of trypsin (4 mg/mL) or 20. Mu.g of promega enzyme was added;

7) Enzymolysis: shaking reaction at 37 ℃ for overnight;

8) Desalting (HLB column purification): the reaction was stopped by standing at room temperature and stopping the enzymatic hydrolysis with 10. Mu.L of 0.5% FA (formic acid). All enzymatic solutions were loaded on a solid phase extraction column sequentially activated with acetonitrile and equilibrated HLB (60 mg/3 ml) with 0.5% FA, eluted sequentially with 3.5mL 0.5% FA, then eluted with 3mL70% ACN+0.5% FA, fixed to 500. Mu.L (dissolved with initial mobile phase ratio) after nitrogen blowing, and the supernatant was centrifuged at 10000rpm for 5min for analysis.

2. High resolution instrument conditions for peptide fragment screening

1) Instrument: waters Xevo Q-TOF

2) Chromatographic conditions: chromatographic column: BEH 300C 18,1.7 μm,2.1 x 100mm;

mobile phase and flow rate: 0.3ml/min, gradient see Table 2;

TABLE 2

Sample injection amount: 5. Mu.L;

column temperature: 40 ℃;

3) Mass spectrometry conditions: da Range 50-2000,Collision Energey: low energy6V, ramp High energy:17-45V.

3. The spectrogram, wherein pork TIC is shown in FIG. 1, beef TIC is shown in FIG. 2, horse TIC is shown in FIG. 3, and chicken TIC is shown in FIG. 4.

4. The characteristic peptide fragments are shown in Table 3.

TABLE 3 characterization of peptide fragments

5. Triple four-bar instrument condition

1) Instrument: shimadzu LC-MS/MS 8050 30A

2) Chromatographic conditions:

chromatographic column: BEH 300C 18,1.7 μm,2.1 x 100mm

Mobile phase and flow rate: 0.3ml/min, acetonitrile A (0.1% FA), water B (0.1% FA), gradient see Table 4:

TABLE 4 Table 4

Sample injection amount: 5 mu L

Column temperature: 40 ℃.

3) Mass spectrometry conditions:

ion source parameters: atomizing air flow rate: 3L/min, heating air flow: 10L/min, dry air flow: 10L/min, interface temperature: 300 ℃. DL temperature: 250 ℃; heating block temperature: 400 ℃.

The optimization and setting of mass spectrum conditions are mainly completed by means of Skyline software (open source software developed by MacCoss laboratories of Washington university, USA) which is provided for free to workers who develop quantitative proteomics research worldwide, methods such as construction SRM, MRM, PRM of Skyline software can be utilized to develop works such as targeted proteomics and targeted DDA. Inputting characteristic fragments into skyline software, giving possible parent ions, child ions, collision energy and other relevant parameters, and further optimizing on an instrument to obtain optimal mass spectrum conditions, wherein the optimal mass spectrum conditions are shown in tables 5-8, and the MRM chromatograms of peptide fragments are shown in figures 5-8.

TABLE 5

TABLE 6

TABLE 7

TABLE 8

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5. Qualitative and quantitative determination

1) Mixing pork and beef according to the proportion of 5%, 10%, 20%, 40%, 60% and 80% of pig respectively, performing enzymolysis extraction, processing according to experimental method, and drawing standard curve of each group of quantitative ions by MRM scanning of HPLC-MS/MS. And respectively taking the mixing proportion of the polypeptides as an abscissa and the area of the selected ion peak of the selected polypeptides as an ordinate, and performing curve fitting. And preparing the mixed meat with a certain proportion according to a sample preparation method, and carrying out calculation by taking the measured data into a standard curve, thereby obtaining the mixing proportion of each meat. The spectrogram and standard curve are shown in figure 9, the mass spectrogram of each peptide fragment is shown in figures 10-18, the chromatograms are shown in figures 19-24, and the standard curve of the peptide fragment is shown in figures 25-28.

2) Quantitative analysis:

according to the sample preparation method, the polypeptide solutions of beef and pork respectively prepared are mixed according to the proportion of 5.0, 10.0, 20.0, 40.0, 60.0 and 80.0 percent of pork respectively, and the ions in table 5 are respectively qualitatively and quantitatively carried out by utilizing the MRM scanning method. The standard curve shows that the selected ions have good linear relation, the linear correlation coefficient of the selected ions reaches more than 0.99, the lowest possible pork is quantified to be 0.5 percent adulterated, and the sensitivity is high.

Claims (5)

1. A method for identifying meat adulterated components by utilizing a high performance liquid chromatography mixed labeling small peptide sample adding method is characterized by adopting a bioinformatics technology and combining a high resolution mass spectrum technology to screen characteristic peptide fragments, and specifically comprises the following steps:

1) Extracting protein in meat and measuring the protein content in the extracting solution;

2) Disulfide bond reduction;

3) Sulfhydryl alkylation;

4) Removing unreacted IAA;

5) Diluting;

6) Performing enzymolysis;

7) Purifying and desalting by using an HLB small column.

2. The method for identifying meat adulterated components by utilizing high performance liquid chromatography mixed labeling small peptide sample adding method as claimed in claim 1, wherein the protein extraction in the meat in step 1) is carried out by extracting, homogenizing, vortex or/and centrifuging, and the supernatant is treated by a filter membrane to obtain the liquid to be tested.

3. The method for identifying meat adulterated components by utilizing high performance liquid chromatography mixed labeling small peptide sample adding method as claimed in claim 1, wherein disulfide bond reduction reaction occurs by vortex and vibration in the step 2).

4. The method for identifying meat adulterated ingredients by utilizing high performance liquid chromatography mixed labeling small peptide sample adding method as claimed in claim 1, wherein the method is characterized in that the diluted extracting solution in the step 5) is obtained by enzymolysis in the step 6), and the peptide fragment solution after enzymolysis is obtained by shaking enzymolysis.

5. The method for identifying meat adulterated components by utilizing high performance liquid chromatography mixed labeling small peptide sample adding method as claimed in claim 1, wherein the step 7) is characterized in that the desalting is carried out by acetonitrile cleaning, balancing, eluting, nitrogen blowing, constant volume taking supernatant and filtering the supernatant to obtain the liquid to be tested to be analyzed.