CN109709229B - Mass spectrum identification method for Atlantic salmon and rainbow trout - Google Patents
Mass spectrum identification method for Atlantic salmon and rainbow trout Download PDFInfo
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Abstract
The invention discloses a mass spectrum identification method for Atlantic salmon and rainbow trout, which comprises the following steps: a) obtaining the amino acid sequence of the Atlantic salmon; b) obtaining an amino acid sequence of the rainbow trout; c) cutting the protein to be detected into a polypeptide fragment mixture with small molecular weight by using a specific enzymolysis method, detecting the molecular weight and fragment information of each polypeptide in the mixture by using an electrostatic field orbital trap high-resolution mass spectrum full-scanning mode, comparing the information with the previously obtained amino acid sequence, and qualitatively judging whether characteristic peptide segments of Atlantic salmon and rainbow trout exist or not; d) selecting characteristic peptide segments qualitatively identified as Atlantic salmon or rainbow trout, and establishing a multi-reaction monitoring method by using tandem quadrupole mass spectrometry for quantitative analysis. The method utilizes the characteristic peptide segment identification of the protein to realize qualitative identification and adulteration quantitative research of the Atlantic salmon and the rainbow trout at the same time, has the lowest detection limit of 1 percent, high sensitivity, high accuracy and strong anti-interference capability, and can be widely applied to the true and false identification of the Atlantic salmon.
Description
Technical Field
The invention relates to a protein mass spectrum detection method, in particular to a mass spectrum identification method for Atlantic salmon and rainbow trout.
Background
The authenticity and source assessment of seafood is an important component in food assessment and identification. Many fish food is labeled with wrong labels, even inferior fish meat and fake fish meat are labeled with high-quality fish meat, which seriously affects the market transparency and damages the health of consumers. At present, the China aquatic circulation and processing society and the Salmon subcontract member unit commonly pass through the salmon identification standard, the salmon is determined as a general name of fishes in the family Salmonidae, and Atlantic salmon and rainbow trout are both one kind of salmon. This has raised widespread concerns in the industry and consumer challenges.
Although Atlantic salmon and rainbow trout are Salmonidae fishes of the same genus, Atlantic salmon is a famous and precious edible fish which prevails internationally, and contains 2.7% of omega-3 fatty acids (EPA, DPA and DHA), and is effective in reducing cholesterol content in a human body, thereby preventing occurrence of cardiovascular diseases. The Atlantic salmon is rich in astaxanthin which is an antioxidant substance, the astaxanthin is very easy to oxidize at high temperature, and the astaxanthin in the Atlantic salmon can not be damaged only by uncooked food, so that the uncooked Atlantic salmon is always a favorite eating method for consumers. Atlantic salmon is mostly imported, expensive and indistinguishable in flesh color, so that the phenomenon of imitating Atlantic salmon by freshwater rainbow trout appears in the domestic market. Salmon is considered by many consumers to be a marine fish such as Atlantic salmon and not a freshwater fish such as rainbow trout. Although both marine and freshwater fish carry parasites in general, they are distinguished by factors such as osmotic pressure. The osmotic pressure of seawater is high, and is different from the environment in human body, so that the parasites carried by seawater fish can not survive in human body for a long time and can not utilize human body to complete life cycle in most cases. As long as the treatment is proper, the risk of infestation of the parasite by the raw marine fish is relatively low. The growth environment of the freshwater fish carrying parasites is similar to the environment in the human body, and the freshwater fish can be deposited in the human body for a long time and is more harmful. About one third of salmon in domestic cities actually produce inland freshwater lakes such as Qinghai-Tibet plateau. In order to reduce the cost, the merchant has good success, and the behavior of imitating the Atlantic salmon by using the domestic rainbow trout seriously jeopardizes the health of consumption. This time the behavior of the chinese aquatic association defining atlantic salmon and freshwater rainbow trout as "salmon" again brought salmon identification to public sight.
Atlantic salmon and rainbow trout are difficult to distinguish by the human eye because of the similarity in color and texture. How to ensure that the expensive salmon purchased by the consumer is indeed Atlantic salmon woollen. At present, the identification technology of salmon includes near infrared spectrum technology and molecular biological technologies such as PCR-RFLP, AFLP, DNA bar code, fluorescence real-time quantitative PCR and the like. The operation steps of PCR-RFLP and AFLP used reagents harmful to human bodies are extremely complicated and long in time. The species identified by the fluorescence real-time quantitative PCR is fast and accurate, but the cost is higher. With the increase in the sales of salmon, the gaps of imported salmon (Atlantic salmon) are further increased, and the behavior of using freshwater fishes such as rainbow trout to impersonate salmon is gradually increased. In order to ensure the right of awareness and physical health of consumers, a novel proteomics-based mass spectrometry detection method is necessary, the characteristic peptide segments of Atlantic salmon and rainbow trout are determined, and a multi-reaction monitoring method is established for qualitative and quantitative analysis.
Disclosure of Invention
The invention aims to solve the technical problem of providing a mass spectrum identification method for Atlantic salmon and rainbow trout, which has high sensitivity, high accuracy and strong anti-interference capability and can be widely applied to the confirmation and detection of the adulteration of the Atlantic salmon.
The invention adopts the technical scheme that the mass spectrum identification method for the Atlantic salmon and the rainbow trout comprises the following steps: a) obtaining and storing amino acid sequences of Atlantic salmon and rainbow trout; b) cutting the protein to be detected into a polypeptide fragment mixture with small molecular weight by using a specific enzymolysis method, detecting the molecular weight and fragment information of each polypeptide in the mixture by using an electrostatic field orbital trap high-resolution mass spectrum full-scanning mode, and comparing the molecular weight and fragment information with the amino acid sequences in the steps a) and b) to realize the identification of the protein and the polypeptide; c) the characteristic peptide segments of Atlantic salmon and rainbow trout are screened out through the comparative analysis of a basic local comparison search tool and a Uniprot database; d) and (3) verifying the characteristic peptide segments of the Atlantic salmon and the rainbow trout by utilizing triple quadrupole mass spectrometry, and establishing a multi-reaction monitoring method for adulteration quantitative analysis.
The method for identifying the mass spectrum of the Atlantic salmon and the rainbow trout, wherein the step a) downloads and obtains the amino acid sequences of the Atlantic salmon and the rainbow trout from a protein database Uniport, and the steps b) and c) determine whether the characteristic peptide fragments of the Atlantic salmon and the rainbow trout exist in the polypeptide fragment mixture by combining mass spectrum results with analysis software.
The mass spectrum identification method for the Atlantic salmon and the rainbow trout comprises the following steps of a), carrying out enzymolysis by using trypsin, and carrying out qualitative protein confirmation on a full-scanning mass spectrogram obtained by an HPLC-Q/Orbitrap in a data-dependent acquisition mode on a sample after enzymolysis; and d) selecting 5-6 ion pairs for each characteristic peptide to carry out multi-reaction monitoring on the polypeptide mixture, thereby completing the quantitative detection of the protein.
The method for identifying the mass spectrum of the Atlantic salmon and the rainbow trout, wherein the step b) comprises the following steps: b1) weighing 2g of homogenized solid sample, adding 2mL of trifluoroacetic acid solution with the mass percentage concentration of 0.1% and 8mL of trichloromethane solution into a 50 mL centrifuge tube, performing vortex oscillation on a vortex oscillator for 5min, performing chloroform degreasing, centrifuging for 3min at 4500r/min, removing supernatant, repeatedly removing grease for 2 times, and concentrating at room temperature under reduced pressure to be nearly dry; b2) adding 10mL of tris (hydroxymethyl) aminomethane-acid salt solution with the molar concentration of 50mmol/L into a sample, adjusting and controlling the pH value to be 7.2, performing vortex oscillation on a vortex oscillator for 1min, performing water bath oscillation extraction at 60 ℃ for 1h, centrifuging for 5min at 10000r/min, and collecting the supernatant for later use; b3) transferring 200 μ L of the above extractive solution into 1.5mL centrifuge tube with low protein adsorption, adding 150 μ L of 500mmol/L ammonium bicarbonate solution, 10 μ L of dithiothreitol solution with molar concentration of 500mmol/L, mixing, and incubating in 75 deg.C constant temperature water bath for 30 min; b4) cooling to room temperature, adding 30 μ L500 mmol/L iodoacetamide solution, and reacting for 30min in dark place; b5) adding 10 μ L of calcium chloride solution with a molar concentration of 100mmol/L and 50 μ L of bovine trypsin solution, mixing well, and placing in 37 deg.C constant temperature water bath for overnight enzymolysis; b6) add 10. mu.L formic acid and mix well to terminate the reaction.
The method for identifying the Atlantic salmon and the rainbow trout by mass spectrometry, wherein the protein quantitative detection in the step d) comprises the following steps: selecting chromatographic columns of different types, column lengths or inner diameters, and recording the change of chromatographic separation; adjusting the flow velocity, gradient or time parameter of the mobile phase, and recording the change of the separation efficiency; controlling the column temperature and the test solution temperature, and recording the reproducibility of the retention time of the chromatogram aiming at the characteristic peptides of the Atlantic salmon and the rainbow trout; adjusting the position of a spray needle, capillary tube voltage, taper hole voltage, gas flow and temperature of each path and collision energy parameters; and observing the arrangement and mass number range of multi-charge ions generated by the proteolysis product by adopting a full scanning mode.
The mass spectrum identification method for the Atlantic salmon and the rainbow trout further comprises the steps of collecting Atlantic salmon and rainbow trout from various different producing areas to perform qualitative confirmation and quantitative detection of protein characteristic peptides, collecting samples from different sources, and obtaining statistical results.
The method for identifying the mass spectrum of the Atlantic salmon and the rainbow trout, wherein in the step c), if the characteristic peptide segment GGLGSAGPTGPR, GEIGGVGANGPSGPQGGR, IGSGLVQEAAPLVDK or GDPGPGGPQGEPGAVGPAGITGDK is detected, the Atlantic salmon is qualitatively judged to be present in the mixture; and c) if the characteristic peptide segment GDPGPGGPQGEQGVVGPAGISGDK is detected in the step c), qualitatively judging that the rainbow trout characteristic peptide segment exists in the mixture.
Compared with the prior art, the invention has the following beneficial effects: the mass spectrometry identification method for the Atlantic salmon and the rainbow trout can qualitatively determine the characteristic peptide sections of the Atlantic salmon and the rainbow trout, and establishes an MRM method for simultaneously qualitatively identifying and accurately measuring the Atlantic salmon and the rainbow trout, and has high sensitivity with the detection limit of 1 percent; the accuracy is high, the anti-interference ability is strong, and the method can be widely applied to the confirmation and detection of the Atlantic salmon adulteration.
Drawings
FIG. 1 is a schematic diagram of a mass spectrometric detection process of characteristic peptide fragments of Atlantic salmon and rainbow trout of the present invention;
FIG. 2 is a chromatogram and mass spectrum of a quantitative ion pair and a qualitative ion pair of characteristic peptide fragments of Atlantic salmon according to the invention;
FIG. 3 is a chromatogram and mass spectrum diagram of the quantitative ion pair and the qualitative ion pair of the rainbow trout characteristic peptide fragment of the invention.
Detailed Description
The invention is further described below with reference to the figures and examples.
FIG. 1 is a schematic diagram of the mass spectrometric detection process of characteristic peptide fragments of Atlantic salmon and rainbow trout of the present invention.
Referring to fig. 1, the detection method adopted by the present invention is mainly a liquid chromatography-mass spectrometry combined method, and mass spectrometry combined electrospray ionization (ESI) is the most reliable polypeptide detection technique so far. The mass spectrum identification method for the Atlantic salmon and the rainbow trout provided by the invention comprises the following steps:
s1) obtaining and storing the amino acid sequences of Atlantic salmon and rainbow trout;
s2) cutting the protein to be detected into a polypeptide fragment mixture with small molecular weight by using a specific enzymolysis method, detecting the molecular weight and fragment information of each polypeptide in the mixture by using an electrostatic field orbital trap high-resolution mass spectrum full-scan mode, and comparing the molecular weight and fragment information with the amino acid sequence in the step S1) to realize the identification of the protein and the polypeptide;
s3) comparing and analyzing a basic local alignment search tool (Blast) with a Uniprot database, and screening out characteristic peptide segments of the Atlantic salmon and the rainbow trout;
s4) utilizing triple quadrupole mass spectrometry to verify the characteristic peptide segments of the Atlantic salmon and the rainbow trout, and establishing a multi-reaction monitoring method for adulteration quantitative analysis. The invention firstly cuts protein into polypeptide fragment mixture with small molecular weight by using a specific enzymolysis or chemical hydrolysis method, detects the molecular weight and fragment information of each polypeptide in the mixture by using an electrostatic field orbit trap high resolution mass spectrum full scan mode, inputs the obtained data of a series of polypeptide molecular weight or fragment ions into a database, searches the known protein corresponding to the data, and realizes the identification of the protein, namely, Peptide Mass Fingerprint (PMF) or peptide fragment ion identification (PFI). And then selecting the characteristic peptide section meeting the requirement according to the detection result and related proteomics software, and establishing a multi-reaction monitoring (MRM) method for quantitative analysis.
The enzyme cutting sites of the trypsin are K and R, after the Atlantic salmon and the rainbow trout are subjected to enzymolysis by the trypsin, GGLGSAGPTGPR, GEIGGVGANGPSGPQGGR, IGSGLVQEAAPLVDK, GDPGPGGPQGEPGAVGPAGITGDK four Atlantic salmon characteristic peptide segments and GDPGPGGPQGEQGVVGPAGISGDK one rainbow trout characteristic peptide segment are obtained, and the total five characteristic peptide segments are five and have different molecular weights, so that the detection by a mass spectrometry method can be used for determining the Atlantic salmon and the rainbow trout respectively.
The mass spectrum identification method for the Atlantic salmon and the rainbow trout provided by the invention is combined with a high performance liquid chromatography-tandem quadrupole mass spectrometry (QqQ) system and Skyline software to establish an MRM method, and can simultaneously realize qualitative identification and quantitative research of the Atlantic salmon and the rainbow trout. The specific establishing process of the characteristic peptide fragment and the MRM method is as follows:
(1) downloading the amino acid sequences of Atlantic salmon and rainbow trout from a Uniport database, scanning the polypeptide mixture generated by enzymolysis of Atlantic salmon and rainbow trout, confirming the protein by using protein search software, and determining the amino acid sequences of the product fragments.
(2) And (3) determining characteristic peptide segments of the Atlantic salmon and the rainbow trout according to a selection principle of characteristic peptides by using Skyline software in combination with a mass spectrum detection result, and selecting 5-6 ion pairs with highest response aiming at each characteristic peptide for establishing an MRM method of a polypeptide mixture, wherein the MRM method is shown in the following table (the x represents quantitative ions), and the rest of the ion pairs are used for assisting qualitative analysis.
The mass spectrum identification method for the Atlantic salmon and the rainbow trout provided by the invention establishes a chromatographic mass spectrum method for simultaneously detecting the Atlantic salmon and the rainbow trout.
(1) Optimizing HPLC conditions: investigating the influence of chromatographic column parameters including type, column length, inner diameter and the like on chromatographic separation; optimizing the influence of parameters such as flow velocity, gradient and time of the mobile phase on the separation efficiency; selecting proper column temperature and test solution temperature; the reproducibility of the chromatographic retention time was examined for characteristic peptide fragments of Atlantic salmon and rainbow trout.
(2) Optimizing ESI-QqQ conditions: optimizing the position of a spray needle, capillary tube voltage, taper hole voltage, gas flow and temperature of each path and collision energy parameters; and observing the arrangement and mass number range of multi-charge ions generated by the proteolysis product by adopting a full scanning mode.
(3) Collecting Atlantic salmon and rainbow trout in various production places, and performing qualitative confirmation and quantitative detection on the Atlantic salmon characteristic peptide segment and the rainbow trout characteristic peptide segment by adopting an optimized pretreatment and chromatographic mass spectrometry method; and collecting qualitative and quantitative data of samples from different sources to obtain statistical results. And (3) inspecting indexes such as linear range, sensitivity, reproducibility and recovery rate of the method.
The mass spectrum identification method for the Atlantic salmon and the rainbow trout provided by the invention comprises the following specific enzymolysis processes:
1) weighing 2g of the homogenized solid sample, adding 2mL of 0.1% trifluoroacetic acid solution and 8mL of trichloromethane solution into a 50 mL centrifuge tube, performing vortex oscillation on a vortex oscillator for 5min, performing chloroform degreasing, centrifuging at 4500r/min for 3min, removing supernatant, repeatedly removing grease for 2 times, and concentrating at room temperature under reduced pressure to be nearly dry.
2) Adding 10mL of tris (hydroxymethyl) aminomethane-acid salt solution (pH 7.2) with the molar concentration of 50mmol/L into a sample, performing vortex oscillation on a vortex oscillator for 1min, performing water bath oscillation at 60 ℃ for extraction for 1h, centrifuging at 10000r/min for 5min, and collecting the supernatant for later use.
3) Accurately transferring 200 μ L of the above extractive solution into 1.5mL centrifuge tube with low protein adsorption, adding 150 μ L of 500mmol/L ammonium bicarbonate solution and 10 μ L of dithiothreitol solution with molar concentration of 500mmol/L, mixing, and incubating in 75 deg.C constant temperature water bath for 30 min.
4) Cooling to room temperature, adding 30 mu L iodoacetamide solution with the molar concentration of 500mmol/L, and reacting for 30min in a dark place; .
5) Adding 10 μ L of 100mmol/L calcium chloride solution and 50 μ L bovine trypsin solution, mixing well, and placing in 37 deg.C constant temperature water bath for enzymolysis overnight.
6) Adding 10 mu L formic acid, mixing evenly and stopping the reaction
7) And carrying out qualitative protein confirmation on the sample subjected to enzymolysis by using a full-scanning mass spectrogram obtained by an HPLC-Q/Orbitrap (data-dependent acquisition, DDA) mode.
8) The protein of the sample after enzymolysis is quantitatively detected on HPLC-QqQ in MRM mode.
In conclusion, the mass spectrometry identification method for the Atlantic salmon and the rainbow trout provided by the invention has high sensitivity, and when the method is used for adulteration detection of the rainbow trout in the Atlantic salmon, the lowest detection limit can reach 1%. The accuracy is high, the anti-interference ability is strong, and the method can be widely applied to the confirmation and detection of the Atlantic salmon adulteration; the chromatogram mass spectra of the Atlantic salmon characteristic peptide fragment quantitative ion pair and the qualitative ion pair are shown in figure 2, the chromatogram mass spectra of the rainbow trout characteristic peptide fragment quantitative ion pair and the qualitative ion pair are shown in figure 3, the abscissa is time (minutes), and the ordinate is mass spectrum intensity.
Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. A mass spectrum identification method for Atlantic salmon and rainbow trout is characterized by comprising the following steps:
a) obtaining and storing amino acid sequences of Atlantic salmon and rainbow trout;
b) adding the solid sample into a trifluoroacetic acid solution with the mass percentage concentration of 0.1% and a trichloromethane solution, carrying out chloroform degreasing, removing supernate, repeatedly degreasing for 2 times, and concentrating under reduced pressure until the solid sample is nearly dry; adding a trihydroxymethyl aminomethane-acid salt solution with the molar concentration of 50mmol/L into a sample, adjusting and controlling the pH value to be 7.2, and extracting supernatant; cutting the protein to be detected in the supernatant into a polypeptide fragment mixture with small molecular weight by using a specific enzymolysis method, detecting the molecular weight and fragment information of each polypeptide in the mixture by using an electrostatic field orbital trap high-resolution mass spectrum full-scanning mode, and comparing the molecular weight and fragment information with the amino acid sequence in the step a) to realize the identification of the protein and the polypeptide;
c) the characteristic peptide segments of Atlantic salmon and rainbow trout are screened out through the comparative analysis of a basic local comparison search tool and a Uniprot database;
d) verifying the characteristic peptide segments of the Atlantic salmon and the rainbow trout by using a triple quadrupole mass spectrometry, determining the characteristic peptide segments of the Atlantic salmon to be GGLGSAGPTGPR, GEIGGVGANGPSGPQGGR, IGSGLVQEAAPLVDK and GDPGPGGPQGEPGAVGPAGITGDK, determining the characteristic peptide segment of the rainbow trout to be GDPGPGGPQGEQGVVGPAGISGDK, and selecting 5-6 ion pairs for each characteristic peptide to perform multi-reaction monitoring on a polypeptide mixture so as to finish qualitative identification and quantitative detection of proteins.
2. The method for mass spectrometric identification of Atlantic salmon and rainbow trout according to claim 1, wherein step a) is performed by downloading the amino acid sequences of Atlantic salmon and rainbow trout from the protein database Uniport, and wherein steps b) and c) are performed by mass spectrometric results in combination with analysis software to determine the presence or absence of characteristic peptide fragments of Atlantic salmon or rainbow trout in the mixture of polypeptide fragments.
3. The method for mass spectrometric identification of Atlantic salmon and rainbow trout according to claim 1, wherein step b) is performed by enzymatic hydrolysis with trypsin, and the protein qualitative confirmation is performed on the full scan mass spectrum obtained by data-dependent acquisition on HPLC-Q/Orbitrap.
4. The method for mass spectrometric identification of Atlantic salmon and rainbow trout of claim 3, wherein said step b) comprises:
b1) weighing 2g of homogenized solid sample, adding 2mL of trifluoroacetic acid solution with the mass percentage concentration of 0.1% and 8mL of trichloromethane solution into a 50 mL centrifuge tube, performing vortex oscillation on a vortex oscillator for 5min, performing chloroform degreasing, centrifuging for 3min at 4500r/min, removing supernatant, repeatedly removing grease for 2 times, and concentrating at room temperature under reduced pressure to be nearly dry;
b2) adding 10mL of tris (hydroxymethyl) aminomethane-acid salt solution with the molar concentration of 50mmol/L into a sample, adjusting and controlling the pH value to be 7.2, performing vortex oscillation on a vortex oscillator for 1min, performing water bath oscillation extraction at 60 ℃ for 1h, centrifuging for 5min at 10000r/min, and collecting the supernatant for later use;
b3) transferring 200 mu L of the extracting solution into a 1.5mL centrifuge tube with low protein adsorption, adding 150 mu L of 500mmol/L ammonium bicarbonate solution and 10 mu L of 500mmol/L dithiothreitol solution, uniformly mixing, and incubating in a constant-temperature water bath kettle at 75 ℃ for 30 min;
b4) cooling to room temperature, adding 30 mu L iodoacetamide solution with the molar concentration of 500mmol/L, and reacting for 30min in a dark place;
b5) adding 10 μ L of calcium chloride solution with a molar concentration of 100mmol/L and 50 μ L of bovine trypsin solution, mixing well, and placing in 37 deg.C constant temperature water bath for overnight enzymolysis;
b6) add 10. mu.L formic acid and mix well to terminate the reaction.
5. The method for mass spectrometric identification of Atlantic salmon and rainbow trout of claim 3, wherein said step d) of qualitative identification and quantitative detection of protein comprises:
selecting chromatographic columns of different types, column lengths or inner diameters, and recording the change of chromatographic separation;
adjusting the flow velocity, gradient or time parameter of the mobile phase, and recording the change of the separation efficiency;
controlling the column temperature and the test solution temperature, and recording the reproducibility of the retention time of the chromatogram aiming at the characteristic peptides of the Atlantic salmon and the rainbow trout;
adjusting the position of a spray needle, capillary tube voltage, taper hole voltage, gas flow and temperature of each path and collision energy parameters; and observing the arrangement and mass number range of multi-charge ions generated by the proteolysis product by adopting a full scanning mode.
6. The method of mass spectrometric identification of Atlantic salmon and rainbow trout of claim 3, further comprising collecting Atlantic salmon and rainbow trout from various origins for qualitative confirmation and quantitative detection of protein-specific peptide fragments, and collecting samples from different sources to obtain statistical results.
7. The method of mass spectrometric identification of Atlantic salmon and rainbow trout of claim 3, wherein the Atlantic salmon is qualitatively determined to be present in the mixture if the characteristic peptide segment GGLGSAGPTGPR, GEIGGVGANGPSGPQGGR, IGSGLVQEAAPLVDK or GDPGPGGPQGEPGAVGPAGITGDK is detected; if the characteristic peptide segment GDPGPGGPQGEQGVVGPAGISGDK is detected, qualitatively judging that the rainbow trout characteristic peptide segment exists in the mixture.
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