CN115825255A - Bird nest authenticity identification method based on characteristic peptide fragment - Google Patents

Abstract

The invention relates to a bird's nest authenticity identification method based on a characteristic peptide fragment. When the authenticity of the edible bird's nest is identified, the edible bird's nest components in the product to be detected are identified by identifying whether 5 characteristic peptide segment sequences related to the edible bird's nest are detected or not through the steps of protein extraction, protein extraction enzyme digestion, edible bird's nest peptide segment UPLC-QTOF-MS detection, characteristic peptide segment detection and the like. The method is used for detecting the authenticity of the bird's nest based on the 5 characteristic peptide fragments, and is high in reliability.

Description

Bird nest authenticity identification method based on characteristic peptide fragment

Technical Field

The invention relates to the technical field of molecular biology, in particular to a method for identifying the authenticity of a bird's nest based on a characteristic peptide fragment.

Background

Edible bird's nest is a nest formed by mixing and coagulating saliva secreted by six kinds of birds of swiftlet of delphiniaceae in the breeding process with feather and the like. Meanwhile, nest holes of some birds of the genus Delphinium of the family Delphiniaceae can also be used as bird nests. The edible bird's nest has a long history in China and is recorded in the Tang dynasty. Until now, people have eaten the cubilose as a high-grade health care product.

From the aspect of components, the cubilose mainly contains protein, carbohydrate, lipid, some inorganic elements, vitamins, hormone and the like. The amino acids contained in the bird's nest are analyzed by research, and the bird's nest is found to contain 20 common amino acids such as aspartic acid, glutamic acid and the like, does not contain hydroxyproline and sarcosine, and contains 8 essential amino acids. The content of saccharides in nidus Collocaliae is second to protein, and mainly contains sialic acid, mannose, glucosamine, galactosamine, etc. The lipid accounts for about 0.14-0.28% of nidus Collocaliae, and the fatty acid components include stearic acid, palmitic acid, linolenic acid, linoleic acid, etc. The edible bird's nest has high inorganic element content and contains trace elements essential for human. The research shows that the higher content elements in the cubilose are four major elements of Ca, mg, na and K, and five necessary trace elements of Fe, zn, mn, cr and Cu.

However, the bird's nest itself is a complex system, and despite years of research, the material basis of the bird's nest is still not clear enough, and counterfeiting or suboptimal events occur frequently. In 2011, food safety accidents that a large amount of nitrite is generated due to the counterfeiting of blood swallows occur in China, so that serious attack is brought to the cubilose industry, and the problem that the quality and the safety of the cubilose cannot be guaranteed to a great extent is also exposed. More attention is paid to the identification and quality control research of the edible bird's nest. With the development of various analytical techniques and molecular biology, the research on edible bird's nest is more intensive.

At present, researchers have developed various methods for identifying the authenticity of the bird's nest, and the bird's nest can be qualitatively identified by using ultraviolet lamp irradiation and specific chemical reaction. And various instrument analysis techniques can also be used for identifying the bird's nest, such as infrared spectroscopy, high performance liquid chromatography and the like. The development of molecular biology provides a new technology for the research of the cubilose, and the molecular biology technology can be used for analyzing the cubilose and identifying the species source of the cubilose. The techniques mainly used for bird's nest analysis include real-time fluorescence PCR and two-dimensional electrophoresis.

Therefore, it is urgently needed to develop a method for identifying characteristic peptide fragments based on characteristic peptide fragments, and simultaneously, the bird's nest and adulterants thereof can be distinguished.

Disclosure of Invention

In order to overcome the technical defects, the main technical purpose of the invention is to provide a method for identifying the authenticity of the bird's nest based on a characteristic peptide fragment, which comprises the following steps:

(1) Preparation of protein samples: adding urea into the protein to be detected, uniformly mixing, performing ice bath ultrasound, uniformly mixing, extracting supernatant, adding cold acetonitrile with the volume 5 times that of the supernatant, and precipitating; centrifuging the precipitate, removing the supernatant, washing the precipitate with cold acetonitrile, and ultrasonically resuspending the precipitate with urea or Ammonium Bicarbonate (ABC) to prepare a protein sample;

(2) Protein alkylation and cleavage: protein concentration determination is carried out by using a BCA kit, the extracted protein is dissolved in Ammonium Bicarbonate (ABC), and reduction and alkylation are carried out; adding mass spectrum level trypsin for digestion for 4h according to the proportion of 1: digesting for 24 hours by 50; centrifuging, taking supernatant, filtering the supernatant by a filter membrane, and taking a subsequent filtrate;

(3) Detection of bird's nest peptide fragment UPLC-QTOF-MS: separating the peptide fragment by an ACQUITY UPLC BEH C18 chromatographic column; the mobile phase A is aqueous solution containing 0.1 percent of formic acid, and the mobile phase B is acetonitrile containing 0.1 percent of formic acid; mobile phase gradient 0-1.5min%; 1.5-3min; 3-13min; 13 to 13.5min; 13.5-1695in%;

(4) Establishing a standard curve of the characteristic peptide fragment of the cubilose: drawing a standard curve by taking the concentration of each peptide segment in the cubilose raw material sample as a standard concentration; performing LC-MS analysis on each standard curve solution from low to high, and preparing a standard curve by taking the peak area of a reference substance as a vertical coordinate and the concentration of the reference substance as a horizontal coordinate;

(5) Characteristic peptide fragment detection: reading the amount of the polypeptides EM, LL, YP, LA, LE in the test solution from the standard curve; and (3) checking whether the following 5 characteristic peptide sequences are detected or not, and if not, identifying that the sample does not contain the bird's nest component.

Further, the step of extracting the supernatant in the step (1) is to transfer the supernatant after centrifugation for 45min at a rotation speed of 15000r/min at 4 ℃.

Further, step (1) the supernatant was added with 5 volumes of cold acetonitrile, precipitated overnight at-20 ℃ in a refrigerator, and centrifuged at 8000r/min at 4 ℃ for 8min, and the supernatant was discarded.

Further, the extracted protein in step (2) was dissolved in 50mM Ammonium Bicarbonate (ABC) so that the protein concentration was 0.6mg/mL.

Further, 2. Mu.L of Dithiothreitol (DTT), 6. Mu.L of Iodoacetamide (IAA) and 1. Mu.L of Dithiothreitol (DTT) are added in the step (2) respectively to react for 30min, 30min and 15min, and reduction and alkylation are carried out.

Further, the flow rate in the column separation step in step (3) was 0.3mL/min, and the column temperature was 35 ℃.

Further, in the step (4), for the raw materials and the finished products of the cubilose, the sequences of the 5 characteristic peptide segments are detected, and the total content of the sequences in the protein is higher than 1 mu g/mg.

Further, when the standard curve is plotted in the step (4), the standard substance of each peptide fragment is prepared into a solution of 0.15625c,0.3125c,0.625c,1.25c,2.5c,5c,10c,20c, and 40c based on the standard concentration c, and the standard curve is plotted with the concentration of each peptide fragment in the bird's nest raw material sample as the standard concentration.

The invention adopts the technical scheme, is based on proteomics technology, identifies the bird's nest protein and peptide fragments with different molecular weights by using an in-gel enzyme digestion method, selects the characteristic peptide fragments to identify the truth of the bird's nest, and has high accuracy.

Drawings

FIG. 1 is a total ion flow diagram and an extracted ion flow diagram of a characteristic peptide segment of a cubilose based on UPLC-QTOF-MS detection;

FIG. 2 is a flow chart of the bird's nest authenticity identification method based on characteristic peptide fragments;

FIG. 3 is a standard curve of 5 characteristic peptide fragments of bird's nest;

FIG. 4 is an extracted ion flow diagram of 5 characteristic peptide fragments of bird's nest;

FIG. 5 shows the detection results of 5 characteristic peptide fragments in the dry cubilose raw materials Y1-Y16;

FIG. 6 shows the detection results of 5 characteristic peptide segments in stewed cubilose products C1, C2 and J1-J4.

Detailed Description

To further illustrate the present invention, reference is made to the following examples. Specifically, the reagents used in the practice of the present invention are all commercially available products, and the databases used in the practice of the present invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.

To screen for suitable characteristic peptide fragments, the criteria for determining peptide fragment screening were: (1) the length is shorter. Shorter peptide fragments may have better stability. (2) Without missed cutting. Trypsin proteolytically cleaves arginine and lysine into peptide fragments at the C-terminus, which if missed cuts occur, results in a change in the actual content of the peptide fragments. (3) There are fewer post-translational modifications. During the protease cleavage, iodoacetamide is added for alkylation to improve the cleavage efficiency, so that cysteine is modified to influence the analysis. Therefore, peptide fragments without cysteine were selected as much as possible. (4) The peptide fragments come from a plurality of different proteins, so that the richness of the peptide fragment sources is ensured.

According to the above criteria, 8 peptide fragments can be screened from all characteristic peptide fragments of cubilose for subsequent qualitative and quantitative research, wherein the 8 peptide fragments are YEDLAQR (SEQ ID No. 1), YPLITTLK (SEQ ID No. 2), LEGAVIELTR (SEQ ID No. 3), LANWPYGHR (SEQ ID No. 4), LLVGFPTYGR (SEQ ID No. 5), FSSQIHNNGR (SEQ ID No. 6), EPDDVTCSTK (SEQ ID No. 7) and EMMVAAFEQAR (SEQ ID No. 8). Then, three bird's nest samples are randomly mixed and the 7 peptide fragments are detected, as shown in fig. 1, 5 peptide fragments have obvious signal peaks, the 5 bird's nest characteristic peptide fragment information is shown in table 1, and the 5 peptide fragments are used as characteristic identification for identifying the authenticity of the bird's nests.

TABLE 1 information of 5 characteristic peptide segments in bird's nest

Based on the above research results, the flow of the method for identifying authenticity of bird's nest of characteristic peptide fragments provided in this embodiment is shown in fig. 3, and the specific steps are as follows:

preparing a dried cubilose raw material sample (Y1-Y16), a stewed cubilose finished product (C1, C2, J1-J4), adulterated soybean protein (W1), egg white (W2), gelatin (W3), tremella (W4), air bladder (W5) and pigskin (W6). The authenticity identification is carried out according to the following steps.

(1) Protein extraction: grinding a sample into dry powder in liquid nitrogen, weighing 0.15-0.2 g, adding 5mL of 8M urea, mixing uniformly, and performing ice-bath ultrasonic treatment (80% and 6 rounds of 8 times); or weighing 1.5-2g of stewed cubilose product, adding 4mL of 8M urea, mixing uniformly, performing ice bath ultrasonic treatment (80%, 8 rounds of 8 times), mixing uniformly, and taking out 1mL of the mixture to an ep tube. Centrifuging at 15000r/min at 4 deg.C for 45min, and transferring the supernatant to new ep tube. To the supernatant was added 5 volumes of cold acetonitrile and precipitated overnight at-20 ℃ in a refrigerator. Centrifuging at 8000r/min at 4 deg.C for 8min, and discarding the supernatant. The pellet was washed 1 time with cold acetonitrile and resuspended (40%, under 3 rounds 7) ultrasonically with 8M urea or 50mM Ammonium Bicarbonate (ABC).

(2) Extracting protein and enzyme digestion: protein concentration determination was performed using the BCA kit. For each 500uL sample, the protein concentration was made around 0.6mg/mL (dissolved in 50mM ABC); respectively adding 2 μ L Dithiothreitol (DTT), 6 μ L Iodoacetamide (IAA), and 1 μ L Dithiothreitol (DTT) (all at 1M) for reaction for 30, and 15min, and performing reduction and alkylation; adding mass spectrum level trypsin for digestion for 4h according to the proportion (protease: protein, m/m) of 1: digesting for 24 hours by 50; centrifuged at 15000rpm for 30 minutes and the supernatant was collected. Before loading, the sample is centrifuged again under the same conditions, and the supernatant is filtered through a 0.22 μm filter membrane, and the filtrate is transferred to a sample bottle.

(3) Detecting bird's nest peptide segment ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS): chromatographic conditions are as follows: the peptide fragments were separated by means of an ACQUITY UPLC BEH C18 (2.1 mm. Times.100mm, 1.7 um) column. Mobile phase a was an aqueous solution containing 0.1% formic acid and mobile phase B was acetonitrile containing 0.1% formic acid. The flow rate was 0.3mL/min and the column temperature was 35 ℃. Mobile phase gradient 0-1.5min%; 1.5-3min; 3-13min; 13 to 13.5min; 13.5 to 1695in. The loading was 2. Mu.L.

Mass spectral data were acquired by agilent 6560Q-TOF mass spectrometry. Data collection was controlled by MassHunter b.08 software. The mass spectrum parameters in positive ion mode are: gas temperature (Gas temperature) 225 ℃, dry Gas (Drying Gas) 10L/min, nebuLizer (Nebulizer) 25psi, sheath Gas temperature (Sheath Gas temperature) 400 ℃, sheath Gas flow (Sheath Gas flow) 12L/min, vcap 3500V, nozzle Voltage (Nozzle Voltage) 500V, fragmentation Voltage (fragmentation Voltage) 400V, and scan range 20-1700m/z.

(4) Establishing a standard curve of the characteristic peptide fragment of the cubilose and verifying a methodology: taking the concentration of each peptide fragment in the bird's nest raw material sample as a standard concentration c, preparing the standard product of each peptide fragment into solutions of 0.15625c,0.3125c,0.625c,1.25c,2.5c,5c,10c,20c and 40c, and drawing a standard curve. The solutions of the standard curves were analyzed by liquid chromatography-mass spectrometer (LC-MS) from low to high, and the standard curves were prepared using the peak areas of the controls as ordinate and the concentrations of the controls as abscissa, as shown in fig. 2. And respectively adding peptide fragment standard substances with the concentrations of 0.5c, c and 2c into the cubilose raw material sample for recovery rate verification.

For quantitative analysis, the linearity and methodology of these 5 peptides were verified, and the results are shown in Table 2, and their linearity coefficients R2 are 0.9950,0.9954,0.9997,0.9972,0.9990, respectively, showing good linearity. In addition, the detection limit, the quantification limit, the repeatability and the recovery rate of the 5 peptide fragments are verified respectively. Fig. 4 is an extracted ion flow diagram of 5 characteristic peptide fragments of bird's nest, from which it can be seen that the 5 peptide fragments have characteristic peaks clearly distinguished from each adulterant. The result shows that all 5 peptide fragments can be used as characteristic peptide fragments of the cubilose for quantitative research.

TABLE 2 methodological validation of characteristic peptide fragments

(5) And (3) testing the bird's nest and the product thereof: the dried bird's nest raw material samples (Y1-Y16) and the stewed bird's nest finished products (C1, C2, J1-J4) were measured according to the above-described test methods, and the measurement results are shown in FIGS. 5 and 6, respectively. Reading the amount of the polypeptides EM, LL, YP, LA and LE in the sample from the standard curve, and calculating to obtain the polypeptide.

For the bird's nest raw material and the finished product, if 5 peptide fragments in table 1 are not detected, the sample does not contain bird's nest components. For the raw materials and the finished products of the cubilose, 5 peptide fragments are detected, and the total content of the peptide fragments in the protein is higher than 1 mu g/mg (0.1 percent), otherwise, the peptide fragments are not qualified. For the finished product of the cubilose, the total content of 5 peptide fragments in the original solution is 8-10ug/mL or more, which is considered as higher quality.

The invention is based on proteomics technology, utilizes an in-gel enzyme digestion method to identify the bird's nest protein and peptide fragments with different molecular weights, selects characteristic peptide fragments to identify the truth of the bird's nest, and has high accuracy.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, means, methods, or steps.

Claims (8)

1. A method for identifying the authenticity of the bird's nest based on a characteristic peptide fragment is characterized by comprising the following steps:

(1) Preparation of protein samples: adding urea into the protein to be detected, uniformly mixing, performing ice bath ultrasound, uniformly mixing, extracting supernatant, adding cold acetonitrile with the volume 5 times that of the supernatant, and precipitating; centrifuging the precipitate, removing the supernatant, washing the precipitate with cold acetonitrile, and ultrasonically resuspending the precipitate with urea or ammonium bicarbonate to prepare a protein sample;

(2) Protein alkylation and cleavage: protein concentration is measured by using a BCA kit, the extracted protein is dissolved in ammonium bicarbonate, and reduction and alkylation are carried out; adding mass spectrum level trypsin for digestion for 4h according to the proportion of 1: digesting for 24 hours by 50; centrifuging, taking supernatant, filtering the supernatant by a filter membrane, and taking a subsequent filtrate;

(3) Detection of bird's nest peptide fragment UPLC-QTOF-MS: separating the peptide fragment by an ACQUITY UPLC BEH C18 chromatographic column; the mobile phase A is aqueous solution containing 0.1 percent of formic acid, and the mobile phase B is acetonitrile containing 0.1 percent of formic acid; mobile phase gradient 0-1.5min; 1.5-3min; 3-13min; 13 to 13.5min; 13.5-1695in;

(4) Establishing a standard curve of the characteristic peptide fragment of the cubilose: drawing a standard curve by taking the concentration of each peptide segment in the cubilose raw material sample as a standard concentration; performing LC-MS analysis on each standard curve solution from low to high, and preparing a standard curve by taking the peak area of a reference substance as a vertical coordinate and the concentration of the reference substance as a horizontal coordinate;

(5) Characteristic peptide segment detection: reading the amount of the polypeptides EM, LL, YP, LA, LE in the test solution from the standard curve; and (3) checking whether the following 5 characteristic peptide sequences are detected or not, and if not, identifying that the sample does not contain the bird's nest component.

2. The method for identifying the authenticity of the bird's nest based on the characteristic peptide fragment as claimed in claim 1, which is characterized in that: the step of extracting the supernatant in the step (1) is to transfer the supernatant after centrifuging at the rotating speed of 15000r/min at 4 ℃ for 45 min.

3. The method for identifying the authenticity of the bird's nest based on the characteristic peptide fragment as claimed in claim 1, which is characterized in that: adding 5 times volume of cold acetonitrile into the supernatant in the step (1), precipitating at-20 ℃ in a refrigerator overnight, centrifuging at 4 ℃ for 8min at 8000r/min, and then discarding the supernatant.

4. The method for authenticating the bird's nest based on the characteristic peptide fragment as claimed in claim 1, wherein the extracted protein is dissolved in 50mM ammonium bicarbonate in the step (2) so that the concentration of the protein is 0.6mg/mL.

5. The method for identifying the authenticity of the bird's nest based on the characteristic peptide fragment as claimed in claim 1, wherein 2 μ L of dithiothreitol, 6 μ L of iodoacetamide and 1 μ L of dithiothreitol are added in the step (2) respectively to react for 30min, 30min and 15min, and reduction and alkylation are carried out.

6. The method for identifying the authenticity of the bird's nest based on the characteristic peptide fragment as claimed in claim 1, wherein the flow rate in the chromatographic column separation step in the step (3) is 0.3mL/min, and the column temperature is 35 ℃.

7. The method for identifying the authenticity of the bird's nest based on the characteristic peptide fragments as claimed in claim 1, wherein in the step (4), for the bird's nest raw materials and finished products, the sequences of the 5 characteristic peptide fragments are detected, and the total content in the protein is higher than 1 μ g/mg.

8. The method for identifying the authenticity of a bird's nest based on a characteristic peptide fragment as claimed in claim 1, wherein when the standard curve is drawn in step (4), the standard substance of each peptide fragment is prepared into a solution of 0.15625c,0.3125c,0.625c,1.25c,2.5c,5c,10c,20c,40c in accordance with the standard concentration c, and the standard curve is drawn with the concentration of each peptide fragment in the bird's nest raw material sample as the standard concentration.

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