CN116338037A - Method for synchronously and quantitatively confirming multiple allergens in food based on mass spectrometry technology and application of method - Google Patents

Abstract

The invention discloses a method for synchronously and quantitatively confirming multiple allergens in food based on mass spectrometry technology and application thereof, wherein the method can simultaneously carry out qualitative and absolute quantification on 12 allergens; by utilizing the characteristic that the isotope labeled characteristic peptide fragment (heavy-standard peptide fragment) and the target characteristic peptide fragment (light-standard peptide fragment) have the same physicochemical property, the isotope labeled peptide fragment is taken as an internal standard, and the linear relation between the area ratio of the light-standard peptide fragment to the heavy-standard peptide fragment and the allergen content is established, and the internal standard method is used for quantification. And adding the same amount of heavy-duty peptide fragments into the sample during detection, determining whether the sample contains the allergen according to whether the target peptide fragments are detected, and calculating according to the area ratio of the detected light-heavy-duty peptide fragments to obtain the absolute mass of the allergen in the sample. The method starts from the actual application scene of processed food products, screens allergen characteristic peptide fragments with strong mass spectrum responsiveness, can effectively filter peptide fragments influenced by matrix effect and processing process, is suitable for peptide fragments detected by multiple allergens at the same time, and has strong specificity, high sensitivity and strong stability.

Description

Method for synchronously and quantitatively confirming multiple allergens in food based on mass spectrometry technology and application of method

Technical Field

The invention belongs to the technical field of food safety detection, and particularly relates to a method for synchronously and quantitatively confirming multiple allergens in food based on a mass spectrum technology and application thereof.

Background

Food allergy is an abnormal immune response of the body to certain food proteins ingested, and food allergy symptoms are mostly manifested in the gastrointestinal tract, skin, respiratory tract and other parts. Several foreign epidemiological studies have shown that food allergy affects nearly 6-8% of children and 2.5% of adults worldwide, with an increasing number of food allergy patients. In addition, some food allergies seriously affect people's life safety and quality of life, so food allergy has become a food safety problem of great concern. In 1995, the national food and agriculture organization (Food and Agriculture Organization of the United Nations, FAO) and world health organization (World Health Organization, WHO) published eight general classes of common allergies, milk, eggs, fish, seafood, peanuts, nuts (including walnuts, hazelnuts, almonds, cashews, etc.), soy and wheat eight general classes of foods were allergens that caused more than 90% of the foods to be allergic. In 2021, sesame was listed as a major class 9 food allergen.

The results of food allergy prevention and treatment research platforms such as food allergy alliance (Food Allergy and Anaphylaxis Network, FAAN), national institute of allergy and infectious disease (National Institute of Allergy and Infectious Diseases, NIAID) and the like show that avoiding contact with or ingestion of allergens is the only effective way to prevent allergic reactions. In the national standard of GB7718-2011 pre-packaged food Label general rule published in 2012 of China, detection and identification of food allergens are also recommended. Therefore, research and establishment of a high-accuracy allergen detection method is a necessary requirement for supporting implementation of food allergen identification standards in China, avoiding food allergy and guaranteeing public health.

Various techniques are currently used for detecting various potential allergens in foods, such as a DNA-level PCR method, a protein-level enzyme-linked immunosorbent assay (ELISA) method and an immunoblotting technique, but have certain limitations. Isotope dilution mass spectrometry (Isotope dilution mass spectrometry, IDMS) has been identified as the most authoritative method of arbitrating for food safety testing internationally, with high accuracy and precision. The IDMS method utilizes the fact that stable isotope labeled substances to be detected and unlabeled substances have the same physicochemical properties, adopts an ion monitoring mode or a multi-reaction monitoring mode, captures respective abundance values through different ion channels, and further carries out accurate quantification. Because the target substance to be detected and the isotope label thereof are treated before the sample, the whole process from the later chromatographic separation, ionization and mass spectrometry is completed, and the influence degree of all factors in the process is the same, various errors can be reduced to the minimum, and the accuracy is higher.

Since the current detection methods are established based on a single food material, they are not compatible with the actual detection of complex processed food products, as well as the presence of multiple allergens. Especially, processed foods generally contain various food materials and ingredients, and if allergens are required to be detected, detection needs to be carried out in a class-by-class manner, so that the efficiency is low. In addition, the phenomenon of allergen cross-talk (cross-talk) is common due to the common production line and other reasons in the processing process, so that the processed food contains hidden allergen 'hidden allergens', and the content is low, thus being easy to cause missed detection.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for synchronously and quantitatively confirming multiple allergens in food based on a mass spectrometry technology and application thereof.

The technical scheme adopted by the invention is as follows: a method for synchronously and quantitatively confirming a plurality of allergens in food based on mass spectrometry technology, which detects one or more items of the presence, the type and the absolute quantity of the allergens in a sample by utilizing isotope dilution mass spectrometry according to characteristic peptide fragments;

the allergen peptide comprises one or more of hazelnut characteristic peptide, walnut characteristic peptide, almond characteristic peptide, cashew characteristic peptide, wheat characteristic peptide, peanut characteristic peptide, soybean characteristic peptide, sesame characteristic peptide, milk characteristic peptide, egg characteristic peptide, fish characteristic peptide and shrimp characteristic peptide;

the characteristic peptide segment of each allergen protein comprises a light-standard peptide segment and a heavy-standard peptide segment, wherein the heavy-standard peptide segment is obtained by labeling one amino acid in the light-standard peptide segment by an isotope;

firstly, performing isotope dilution mass spectrometry detection on a light-standard peptide fragment and a heavy-standard peptide fragment with known concentrations to obtain a standard curve of the area ratio of the light-standard peptide fragment to the heavy-standard peptide fragment and the concentration ratio of the light-standard peptide fragment to the heavy-standard peptide fragment; and extracting peptide fragments in the food sample, adding the same-quantity heavy-standard peptide fragments for isotope dilution mass spectrometry detection, and comparing the detection result with a standard curve to realize the detection of the allergen in the sample.

Preferably, the allergen peptide fragments comprise at least two of hazelnut-signature peptide fragments, walnut-signature peptide fragments, almond-signature peptide fragments, cashew-signature peptide fragments, wheat-signature peptide fragments, peanut-signature peptide fragments, soybean-signature peptide fragments, sesame-signature peptide fragments, milk-signature peptide fragments, egg-signature peptide fragments, fish-signature peptide fragments, and shrimp-signature peptide fragments;

preferably, the allergen peptide fragments comprise plant-derived hazelnut-characteristic peptide fragments, walnut-characteristic peptide fragments, almond-characteristic peptide fragments, cashew-characteristic peptide fragments, wheat-characteristic peptide fragments, peanut-characteristic peptide fragments, soybean-characteristic peptide fragments and sesame-characteristic peptide fragments; alternatively, the allergen peptide fragments include milk characteristic peptide fragments, egg characteristic peptide fragments, fish characteristic peptide fragments and shrimp characteristic peptide fragments of animal origin;

alternatively, the allergen peptide fragments include hazelnut, walnut, almond, cashew, wheat, peanut, soybean, sesame, milk, egg, fish, and shrimp peptide fragments.

Preferably, the light-standard peptide fragment and the heavy-standard peptide fragment in the hazelnut characteristic peptide fragment are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 or as SEQ ID NO.3 and SEQ ID NO. 4;

the light-standard peptide and the heavy-standard peptide in the walnut characteristic peptide are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6; the light-standard peptide and the heavy-standard peptide in the almond characteristic peptide are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8; the light-standard peptide and the heavy-standard peptide in the cashew nut characteristic peptide are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10;

the light-standard peptide and the heavy-standard peptide in the wheat characteristic peptide are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12;

the light-standard peptide and the heavy-standard peptide in the peanut characteristic peptide are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14;

the light-standard peptide and the heavy-standard peptide in the soybean characteristic peptide are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16;

the light-standard peptide and the heavy-standard peptide in the sesame characteristic peptide are respectively shown as SEQ ID NO.17 and SEQ ID NO. 18;

the light-standard peptide and the heavy-standard peptide in the milk characteristic peptide are respectively shown as SEQ ID NO.19 and SEQ ID NO.20 or as SEQ ID NO.21 and SEQ ID NO. 22;

the light-standard peptide and the heavy-standard peptide in the egg characteristic peptide are respectively shown as SEQ ID NO.23 and SEQ ID NO. 24;

the light-standard peptide and the heavy-standard peptide in the fish characteristic peptide are respectively shown as SEQ ID NO.25 and SEQ ID NO. 26; the light-standard peptide and the heavy-standard peptide in the shrimp characteristic peptide are respectively shown as SEQ ID NO.27 and SEQ ID NO. 28; SEQ ID NO.1 ADIYTEQVGR

SEQ ID NO.2 ADIYTEQV*(13C5,15N)GR

SEQ ID NO.3 TNDNAQISPLAGR

SEQ ID NO.4 TNDNAQISPL*(13C6,15N)AGR

SEQ ID NO.5 ISTVNSHTLPVLR

SEQ ID NO.6 ISTVNSHTLPVL*(13C6,15N)R

SEQ ID NO.7GNLDFVQPPR

SEQ ID NO.8GNLDFV*(13C5,15N)QPPR

SEQ ID NO.9ADIYTPEVGR

SEQ ID NO.10ADIYTPEV*(13C5,15N)GR

SEQ ID NO.11YFIALPVPSQPVDPR

SEQ ID NO.12YFIALPVPSQPV*(13C5,15N)DPR

SEQ ID NO.13RPFYSNAPQEIFIQQGR SEQ ID NO.14RPFYSNAPQEIFIQQGR*(13C6,15N4)

SEQ ID NO.15VLIVPQNFVVAAR

SEQ ID NO.16VLIVPQNFVV*(13C5,15N)AAR

SEQ ID NO.17AFYLAGGVPR

SEQ ID NO.18AFYLAGGV*(13C5,15N)PR

SEQ ID NO.19HQGLPQEVLNENLLR

SEQ ID NO.20HQGLPQEVLNENLL*(13C6,15N)R

SEQ ID NO.21TPEVDDEALEK

SEQ ID NO.22TPEVDDEAL*(13C6,15N)EK

SEQ ID NO.23GGLEPINFQTAADQAR SEQ ID NO.24GGLEPINFQTAADQAR*(13C6,15N4)

SEQ ID NO.25TIDDLEDELYAQK SEQ ID NO.26TIDDLEDELYAQK*(13C6,15N2)

SEQ ID NO.27ALSNAEGEVAALNR SEQ ID NO.28ALSNAEGEVAAL*(13C6,15N)NR。

Preferably, the liquid chromatography conditions are: mobile phase a was 0.1% formic acid/water, mobile phase B was 0.1% formic acid/acetonitrile, flow rate 0.2mL/min; column temperature 40 ℃; the chromatographic column is a C18 chromatographic column; the liquid chromatographic gradients are shown in the following table;

the mass spectrum conditions are as follows: a HESI (electro spray) source positive ion reaction mode and a PRM scanning mode are used; the ion source parameters of atomizing gas, gas curtain gas, auxiliary heating gas and collision gas are high-purity nitrogen or other suitable gases, and the flow of each gas is regulated before use, so that the mass spectrum sensitivity meets the requirement; PRM scan set resolution of 70,000, C-trap maximum capacity of 1X 10 ⁶ The maximum injection time of C-trap is 100ms, the isolation window is 1.6m/z, and the peptide fragments are crushedThe crack impact energy is set to a default value 27;

the inclusion list is set up as follows;

the application of the method for synchronously and quantitatively confirming the multiple allergens in the food based on the mass spectrum technology in the detection of the allergens in the processed food.

Preferably, diluting the stock solution containing the light-standard peptide fragment with a food matrix solution without the light-standard peptide fragment, adding the heavy-standard peptide fragment, detecting by isotope dilution mass spectrometry, and drawing to obtain a standard curve; pretreating a processed food sample to be detected, adding an equivalent weight-marked peptide fragment, performing isotope dilution mass spectrometry detection, and judging whether the processed food sample to be detected contains a corresponding light-marked peptide fragment according to the existence of a characteristic peak;

and/or comparing the characteristic peak area detection result with a standard curve to calculate the concentration of the corresponding light-label peptide segment.

Preferably, in the system for detection, the concentration of the wheat heavy-standard peptide is not lower than 100 fmol/mu L, the concentration of the almond heavy-standard peptide is not lower than 200 fmol/mu L, and the concentration of the heavy-standard peptide of hazelnuts, walnuts, cashew nuts, peanuts, soybeans and sesame is not lower than 50 fmol/mu L.

Preferably, the quantitative standard curves for the characteristic peptide fragments in different matrices are as follows;

wherein Y is the ion peak area ratio of the light-standard peptide fragment/heavy-standard peptide fragment of the tested component, and X is the concentration of the tested component.

Preferably, the allergen content is calculated by formula 1;

wherein:

c, the content of the tested components in the test sample is expressed in milligrams per kilogram (mg/kg);

x-the concentration of the measured component solution obtained from the standard working curve in femtomoles per microliter (fmol/. Mu.L);

v-sample constant volume in milliliters (mL);

m-molecular weight of allergen protein in kilograms per mole (g/mol);

m-sample weight, unit gram (g).

Preferably, the pretreatment process of the processed food sample to be detected comprises the processes of protein extraction, reduction, alkylation, enzymolysis, desalination and suspension in sequence;

the preparation of the food matrix solution sequentially comprises the processes of protein extraction, reduction, alkylation, enzymolysis, desalination and suspension drying.

The invention has the advantages and positive effects that: the method can realize qualitative and quantitative detection at the same time, and can detect one or more allergens of 12 or single allergens at the same time; starting from the processed food product of the practical application scene, the allergen characteristic peptide segment with strong mass spectrum responsiveness is screened, the peptide segment influenced by the matrix effect and the processing process (high temperature, maillard reaction, low pH and the like) and the peptide segment suitable for simultaneous detection of multiple allergens can be effectively filtered, and the finally screened peptide segment has strong specificity, high sensitivity and strong stability.

Drawings

FIG. 1 is a total ion flow diagram of PRM monitoring of individual allergen signature peptides;

FIG. 2 is a chromatogram of the PRM monitoring of hazelnut-TND signature peptides;

FIG. 3 is a chromatogram of PRM monitoring of hazelnut-ADI signature peptides;

FIG. 4 is a chromatogram of PRM monitoring of walnut signature peptide fragments;

FIG. 5 chromatogram of PRM monitoring of almond signature peptide fragment;

FIG. 6 is a chromatogram of a PRM monitoring of cashew nut signature peptides;

FIG. 7 chromatogram of sesame signature peptide PRM monitoring;

FIG. 8 chromatogram of PRM monitoring of peanut signature peptide fragments;

FIG. 9 chromatogram of soybean characteristic peptide PRM monitoring;

FIG. 10 chromatogram of PRM monitoring of wheat signature peptide fragment;

FIG. 11 milk-B5 characteristic peptide PRM detection chromatogram;

FIG. 12 milk-B9 characteristic peptide PRM detection chromatogram;

FIG. 13 PRM detection chromatogram of the chicken egg characteristic peptide fragment;

FIG. 14 PRM detection chromatogram of fish signature peptide fragment;

FIG. 15 PRM detection chromatogram of shrimp signature peptide fragment;

FIG. 16 is a standard curve of product ion peak area integration and quantification of hazelnut-TND signature peptides in chocolate matrix;

FIG. 17 is a standard curve of product ion peak area integration and quantification of hazelnut-ADI signature peptide fragments in chocolate matrix;

FIG. 18 integration of product ion peak area of walnut signature peptide fragments in chocolate matrix and quantification of standard curves;

FIG. 19 shows product ion peak area integration and quantification of almond feature peptide fragments in chocolate matrix standard curve;

FIG. 20 is a graph of product ion peak area integration and quantification of cashew characterizing peptide segments in a chocolate matrix;

FIG. 21 integration of product ion peak area of sesame signature peptide fragments in chocolate matrix and quantification of standard curves;

FIG. 22 product ion peak area integration and quantification of peanut signature peptide fragments in chocolate matrix standard curve;

FIG. 23 product ion peak area integration and quantification of soy signature peptide fragments in chocolate matrix standard curve;

figure 24 product ion peak area integration and quantification of wheat signature peptide fragments in chocolate matrix standard plots.

FIG. 25 integration of product ion peak area and quantification of milk-B5 characteristic peptide fragments in flour matrix standard curve;

FIG. 26 integration of product ion peak area and quantification of milk-B9 signature peptides in flour matrix standard curve;

FIG. 27 product ion peak area integration and quantification of characteristic peptide fragments of eggs in flour matrix standard curve;

FIG. 28 integration of product ion peak area of fish characteristic peptide fragments in ham sausage matrix and quantification of standard curve;

FIG. 29 integration of product ion peak area of shrimp signature peptide fragments in ham sausage matrix and quantification of standard plots.

Detailed Description

Embodiments of the present invention are described below with reference to the accompanying drawings.

The invention discloses a method for synchronously and quantitatively confirming multiple allergens in food based on mass spectrometry technology and application thereof, wherein the method can simultaneously carry out qualitative and/or quantitative detection on 12 or one or more allergens detected independently, 8 plant-source allergens detected independently or 4 animal-source allergens detected independently, plant-source allergens and animal-source allergens can be detected simultaneously, and particularly, one or more of hazelnut, walnut, almond, cashew, wheat, peanut, soybean, sesame, milk, egg, fish and shrimp allergens can be detected simultaneously. By the detection method, which allergic substance is contained in the object to be detected can be obtained or known, and meanwhile, the absolute content of the allergic substance is calculated. Is especially suitable for detecting allergen in processed food with various raw materials.

The characteristic peptide fragments required for detection comprise light-standard peptide fragments and heavy-standard peptide fragments, wherein the light-standard peptide fragments correspond to allergen/sensitization protein specific peptide fragments of different foods, and the heavy-standard peptide fragments are obtained by labeling one amino acid in the light-standard peptide fragments by isotopes. The method utilizes the characteristic that the isotope labeled characteristic peptide fragment (the heavy standard peptide fragment) and the target characteristic peptide fragment (the light standard peptide fragment) have the same physicochemical property, takes the isotope labeled peptide fragment as an internal standard, establishes the linear relation between the area ratio of the light standard peptide fragment to the heavy standard peptide fragment and the concentration ratio of the light standard peptide fragment to the heavy standard peptide fragment, and carries out quantification by an internal standard method. And during detection, adding the same amount of heavy-standard peptide into the peptide extract of the food sample, determining whether the target peptide contains the allergen according to whether the target peptide is detected, and calculating according to the area ratio of the detected light-heavy-standard peptide to obtain the absolute mass of the allergen in the sample. In the implementation, isotope dilution mass spectrometry detection is carried out on a light-standard peptide fragment and a heavy-standard peptide fragment with known concentration, so that a standard curve of the area ratio of the light-standard peptide fragment to the heavy-standard peptide fragment to the concentration ratio of the light-standard peptide fragment to the heavy-standard peptide fragment is obtained; and extracting peptide fragments in the food sample, adding the same-quantity heavy-standard peptide fragments for isotope dilution mass spectrometry detection, and comparing the detection result with a standard curve to realize the detection of the allergen in the sample.

The light standard peptide fragments are shown in tables 1-1 and 1-2, and are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO. 27; the heavy standard peptide fragments are shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26 and SEQ ID NO. 28.

Detection of processed food product allergens is a major and difficult aspect of food allergen monitoring. The key point is that the sensitized substance is not easy to distinguish due to processing into different forms, and has 'concealment'. The difficulty is that food products often have cross-contamination during processing due to common processing lines, common storage spaces, etc., which necessarily introduce "trace" amounts of food allergens. In addition, processed food products often contain a variety of sensitizers, such as nut breads, and may contain simultaneously a variety of sensitizers such as wheat, eggs, milk, nuts, and the like. Moreover, food processing often undergoes high temperature, fermentation, etc. processes, and because of the complexity of the food ingredients, there is a matrix effect. The complexity of the above various food samples places high demands on qualitative and quantitative detection of food allergens.

The research starts from the actual application scene of 'processed food products' (building-unit), screens sensitive, specific and efficient allergen characteristic peptide fragments, and is different from the peptide fragment screening strategy starting from single food sensitization substances in the prior art, and the screening strategy is closer to the application scene, so that peptide fragments influenced by matrix effect and processing processes (high temperature, maillard reaction, low pH and the like) and peptide fragments suitable for simultaneous detection of multiple allergens can be effectively filtered, and the screening efficiency and speed are greatly improved. Meanwhile, the peptide fragment is ensured to have the characteristics of higher sensitivity, accuracy, high reproducibility and high flux in the actual application scene, namely, the detection of commercial processed food products.

In use, the target peptide (light-chain peptide) is prepared from peptide fragments without isotope labeled amino acid by preparing 10 from 0.1% formic acid solution ⁶ The fmol/mu L stock solution is divided into 10 mu L for each tube, and frozen at-80 ℃ for a long time; the internal standard peptide fragment (re-standard peptide fragment) is the corresponding peptide fragment containing isotopically labeled amino acid, and the re-standard peptide fragment is prepared into 10 by using 0.1% formic acid solution ⁶ The stock solution of fomol/. Mu.L was dispensed into 10. Mu.L each tube and frozen at-80℃for a long period. In some embodiments of the invention, the final wheat concentration of the heavy standard is not lower than 100 fmol/mu L, the almond concentration of the heavy standard peptide is not lower than 200 fmol/mu L, and the other concentration of the heavy standard peptide is not lower than 50 fmol/mu L by combining the detection cost and the detection accuracy.

The substrate solution does not comprise target peptide segments, products such as flour, chocolate, ice cream, oatmeal, biscuits, breakfast cereal powder and the like are purchased from supermarkets, the ingredients contained in the ingredients table are referred to, total proteins are extracted and subjected to enzymolysis, mass spectrum scanning is carried out, and the protein ingredients contained in the products are confirmed by adopting a full MS-ddMS2 scanning mode. Reference is made to the matrices required for AOAC SMPR 2016.002 and matrices without target protein can be used for peptide fragment dilution. Diluting the stock solution containing the light-standard peptide by using a food matrix without the light-standard peptide, taking the heavy-standard peptide as an internal standard, establishing a linear relation between the area ratio of the light-standard peptide to the heavy-standard peptide and the allergen content, and drawing a standard curve.

The amino acid sequence, charge number, charge-to-mass ratio (m/z) of the precursor ion and the product ion of the plant-derived peptide fragment are shown in Table 1-1;

TABLE 1-1

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The amino acid sequence, the charge number, the charge-to-mass ratio (m/z) of the precursor ions and the product ions and the like of the animal-derived peptide fragment are shown in tables 1-2;

TABLE 1-2

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The preparation of the standard curve and the detection process of the sample to be detected need to adopt the same isotope dilution mass spectrometry detection condition.

In the detection process of the invention, various types of liquid phase-mass spectrometers can be used; the following detection conditions may be employed in certain embodiments of the present invention. Liquid chromatography conditions:

instrument: thermo science ^TM Vanquish Binary Flex UHPLC or equivalent. Wherein Thermo science ^TM The UHPLC model Vanquish Binary Flex UHPLC comprises the following components: system Base Vanquish Flex (P/N VF-S01-A); binary Pump F (P/N VF-P10-A-01); split sample FT (P/N VF-A10-A); column Compartment H (P/N VH-C10-A); MS Connection Kit Vanquish (P/N6720.0405); vanquish F Pumps 100. Mu.L Mixer Set (P/N6044.5100); vanquish Split Sampler HT Sample Loop, 100. Mu.L (P/N6850.1913).

Separation conditions: mobile phase a:0.1% formic acid/water, mobile phase B:0.1% formic acid/acetonitrile;

chromatographic column: shim-packGISS-HP C18 (metal free column) 3.0 μm,2.1 mm. Times.150 mm (P/N: 227-30924-03);

column temperature: still air at 40 ℃;

the liquid chromatographic gradients are shown in table 2;

TABLE 2 high performance liquid chromatography gradient elution procedure

Mass spectrometry conditions:

mass spectrometry instrument: thermo science ^TM Q Exactive

Mass spectrometry source parameters are shown in table 3;

table 3 scanning selected mass spectrometry source parameters

Sheath gas flow rate	35
		Aux gas flow rate	10
Sweep gas flow rate	0
		Spray voltage	3.8kV
Capillary temp	320℃
		S-lens RF level	55.0
Aux gas heater temp	350℃

Scanning mode: PRM (PRM)

Scanning conditions: as shown in table 4;

TABLE 4-1 Properties of the method

TABLE 4-2 Properties of PRM

The inclusion list settings are shown in Table 5;

TABLE 5

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The mixed solution of the light-label peptide fragment and the heavy-label peptide fragment can be detected by the detection conditions, or the mixture of the light-label peptide fragment extract and the heavy-label peptide fragment can be detected, as shown in fig. 1, which is a total ion flow chart of the PRM monitoring of each allergen characteristic peptide fragment in table 1, ion peaks of different allergic substances can be clearly shown and separated, and fig. 2-15 are chromatograms of the PRM monitoring of each characteristic peptide fragment, wherein the corresponding light-label peptide fragment and the heavy-label peptide fragment have the same peak-out time and similar peak types. And detecting the light-standard peptide sample with gradient concentration, quantifying the data processing flow by using Xcalibur software of a QE mass spectrometer, and carrying out according to an operation instruction to obtain the area of the light-standard peptide and the area of the heavy-standard peptide of each allergen in a matrix, thereby obtaining a standard curve of the corresponding relation between the area ratio and the concentration, and obtaining the lowest quantification limit.

By the above method and detection conditions, it is possible to quantitatively detect whether or not the food sample to be detected contains 12 kinds of allergens/sensitized proteins shown in tables 1-1 and 1-2, and simultaneously. Firstly preparing a standard curve, then preprocessing a food sample to be detected, wherein the preprocessing process sequentially comprises the processes of protein extraction, reduction, alkylation, enzymolysis and desalination, isotope dilution mass spectrometry detection is carried out after a heavy-standard peptide fragment is added, whether the food sample to be detected contains allergic substances corresponding to the light-standard peptide fragment or not is judged, if the retention time of the substances to be detected is within +/-2.5% of the retention time of the heavy-standard peptide fragment, qualitative ions of the peptide fragment selected in the sample are all present (3 product ions of the light-standard peptide fragment in table 1), and the corresponding allergen protein is detected; and comparing the detection result with a standard curve to calculate the concentration of the detected allergic substance, and calculating the content of the allergic substance.

The sample pretreatment method comprises the following steps:

1 protein extraction, reduction, alkylation and enzymolysis

(1) After the food sample was sufficiently ground, 3g was weighed into a 50mL centrifuge tube, 20mL300mM Tris (pH 9.2), 2M urea was added, and the mixture was incubated with shaking at 20℃for 30min and water at 90℃for 10min.

(2) Centrifuge at 5000g for 10min.

(3) 1mL of the supernatant was taken and dissolved in 1mL buffer (200 mM NH) ₃ HCO ₃ pH 8.2) was diluted. In certain embodiments of the invention, 10. Mu.L of the supernatant may also be subjected to SDS-PAGE; the kit protein concentration was quantified with protein. (4) 40. Mu.L of 500mM DTT was added and incubated at 75℃for 30min; 80. Mu.L of 500mM IAA (protected from light) was incubated at room temperature for 30min.

(5) 100. Mu.L of 1% pancreatin acetic acid solution was added, overnight at 37 ℃.

(6) The next day, 3000g was centrifuged for 30 seconds, and the supernatant was incubated at 90℃for 10 minutes to terminate the enzymatic hydrolysis.

(7) Centrifuge 12000g for 30min, collect supernatant (500. Mu.L of supernatant was enough).

2 desalination of

The peptide extract obtained in the above step is desalted using monosain C18 desalting column (GL Sciences inc.) or other equivalent product, see product description. The method is characterized in that:

(1) Adjusting the pH value of a sample: the pH of the sample was adjusted to about 3-4 with formic acid.

(2) condition column: 200. Mu.L of acetonitrile was added and centrifuged at 5000g for 1min. 200. Mu.L of 0.1% formic acid was added and centrifuged at 5000g for 1min.

(3) Loading: the sample is applied to the column and centrifuged at 5000g for 1-2min.

(4) 300. Mu.L of 0.1% formic acid was added and centrifuged at 5000g for 1min.

(5) The column was placed in a recovery tube, 300. Mu.L of 80% acetonitrile (0.1% formic acid) was added, and 5000g was centrifuged for 1-2min.

And centrifuging the obtained solution to obtain the desalted peptide fragment.

3 vacuum suspension

The desalted peptide fragments were suspended by a vacuum concentrator.

The dried peptide was resuspended in 0.1% chromatography-pure formic acid equivalent to the supernatant obtained in step 1- (7) before loading, and 12000g was centrifuged for 30min or passed through a 0.22 μm PES filter. Before mass spectrum scanning, the peptide fragment concentration is determined by using a peptide fragment quantitative kit, and a proper amount of sample is loaded according to mass spectrum requirements.

The method can be used for food sample pretreatment and can also be used for preparing a matrix solution.

The method can realize qualitative and quantitative detection at the same time, and can detect one or more plant-derived allergens of 12 species or independently; from the 'processed food products' of practical application scenes, allergen characteristic peptide fragments with strong mass spectrum responsiveness are screened, peptide fragments influenced by matrix effect and processing process (high temperature, maillard reaction, low pH and the like) can be effectively filtered, and the method has good detection effect in bread, biscuits, cakes, ice cream, yoghurt and the like.

The following description of the present invention is made with reference to the accompanying drawings, wherein the experimental methods without specific description of the operation steps are performed according to the corresponding commodity specifications, and the instruments, reagents and consumables used in the embodiments can be purchased from commercial companies without specific description.

Example 1: standard curve drawing

1.1 each of the peptide fragments (light and heavy) in tables 1-1 and 1-2 were synthesized by commercial peptide fragment synthesis companies, with purity requirements greater than 98%.

1.2 target peptide fragments, also referred to as light-gauge peptide fragments, are each peptide fragment of tables 1-1 and 1-2 that does not contain isotopically labeled amino acids. The target peptide was prepared to 10% with 0.1% formic acid solution ⁶ The fmol/mu L stock solution is divided into 10 mu L for each tube, and frozen at-80 ℃ for a long time; the internal standard peptide, also called the standard peptide, is the corresponding isotopically labeled amino acid containing peptide in tables 1-1 and 1-2. The re-labeled peptide fragments were also formulated to 10 using 0.1% formic acid solution ⁶ fomol/. Mu.L stock.

1.3 preparation of matrix solutions

Taking chocolate matrix as an example;

(1) Selecting a chocolate sample without target peptide fragments, fully grinding, weighing 3g, putting into a 50mL centrifuge tube, adding 20mL300mM Tris (pH 9.2) and 2M urea, oscillating at 20 ℃ for 30min, and bathing at 90 ℃ for 10min.

(2) Centrifuge at 5000g for 10min.

(3) 1mL of the supernatant was taken and dissolved in 1mL buffer (200 mM NH) ₃ HCO ₃ pH 8.2) was diluted. Taking 10 mu L of supernatant, and running SDS-PAGE; the kit protein concentration was quantified with protein.

(4) 40. Mu.L of 500mM DTT was added and incubated at 75℃for 30min; 80. Mu.L of 500mM IAA (protected from light) was incubated at room temperature for 30min.

(5) 100. Mu.L of 1% pancreatin acetic acid solution was added, overnight at 37 ℃.

(6) The next day, 3000g was centrifuged for 30 seconds, and the supernatant was incubated at 90℃for 10 minutes to terminate the enzymatic hydrolysis.

(7) Centrifuging 12000g for 30min, and collecting supernatant;

(8) Desalting the supernatant with monostein C18 desalting column (GL Sciences Inc.) or other equivalent products;

(9) The desalted peptide was suspended by a vacuum concentrator, and the suspended peptide was reconstituted with 0.1% of the same amount of the chromatographic pure formic acid as the supernatant obtained in step (7) to prepare a matrix solution.

According to the steps, matrix solutions of milk matrix, biscuit matrix and other food types can be prepared.

1.4 series of standard solutions preparation:

and (3) taking the storage solution of the 9 light standard peptide fragments, and diluting the storage solution to 2500, 1000, 500, 250, 100, 50, 25, 10,5,2.5,1,0.5 and 0.25 fmol/mu L standard concentration by using the prepared matrix solution without the target peptide fragments. Meanwhile, a fixed amount of heavy standard peptide fragments are added into each tube of light standard working solution with different concentrations, the final concentration of the wheat heavy standard peptide fragments is 100 fmol/mu L, the concentration of the almond heavy standard peptide fragments is 200 fmol/mu L, and the concentrations of the rest heavy standard peptide fragments are 50 fmol/mu L.

1.5. Mu.L of each of the above-prepared serial standard solutions was taken and subjected to isotope dilution mass spectrometry.

1.6 liquid chromatography conditions

Instrument: thermo science ^TM Vanquish Binary Flex UHPLC or equivalent. Wherein Thermo science ^TM The UHPLC model Vanquish Binary Flex UHPLC comprises the following components: system Base Vanquish Flex (P/N VF-S01-A); binary Pump F (P/N VF-P10-A-01); split sample FT (P/N VF-A10-A); column Compartment H (P/N VH-C10-A); MS Connection Kit Vanquish (P/N6720.0405); vanquish F Pumps 100. Mu.L Mixer Set (P/N6044.5100); vanquish Split Sampler HT Sample Loop, 100. Mu.L (P/N6850.1913)

Separation conditions: mobile phase a:0.1% formic acid/water; mobile phase B:0.1% formic acid/acetonitrile;

chromatographic column, shim-packGISS-HP C18 (metal free column) 3.0 μm,2.1mm 150mm (P/N: 227-30924-03);

column temperature: still air at 40 ℃;

the liquid chromatography gradient is shown in Table 2.

1.7 Mass Spectrometry conditions

Mass spectrometry instrument: thermo science ^TM Q exact; mass spectrometry source parameters are as in table 3; the mass spectrum conditions are as follows: a HESI (electro spray) source positive ion reaction mode and a PRM scanning mode are used; the ion source parameters of the atomizing gas, the gas curtain gas, the auxiliary heating gas and the collision gas areHigh-purity nitrogen or other suitable gases, and before use, the flow of each gas is regulated, so that the mass spectrum sensitivity meets the requirements; PRM scan set resolution of 70,000, C-trap maximum capacity of 1X 10 ⁶ The maximum injection time of the C-trap is 100ms, the isolation window is 1.6m/z, and the fragmentation collision energy of the peptide fragment is set to be 27; scanning conditions are as shown in table 4; the inclusion list settings are as in Table 5. The detection result is quantitatively processed by using xcalibur software of a QE mass spectrometer according to an operation instruction, so that the area of a light-standard peptide segment and the area of a heavy-standard peptide segment of each allergen in a matrix are obtained, a standard curve of the corresponding relation between the area ratio and the light-standard concentration is obtained, and the lowest quantitative limit is obtained. As shown in the left side graph of fig. 16-29, the quantitative ion peak condition of the light-label peptide fragment in a detection sample is shown, and as shown in the right side graph of fig. 16-29, the generated standard curve condition is shown; the standard curve generated by different characteristic peptides in different matrices is shown in Table 6.

TABLE 6 quantitative Standard Curve of allergen characterization peptide fragments in different matrices

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Example 2: pretreatment of food samples

2.1 extracting protein from food samples, and sequentially carrying out reduction, alkylation and enzymolysis.

(1) After taking a food sample, fully grinding, weighing 2g, putting into a 50mL centrifuge tube, adding 20mL300mM Tris (pH 9.2) and 2M urea, carrying out shaking warm bath for 30min at 20 ℃ and carrying out water bath for 10min at 90 ℃.

(2) Centrifuge at 5000g for 10min, 1mL of supernatant was taken and dissolved in 1mL buffer (200 mM NH ₃ HCO ₃ pH 8.2) was diluted. 10. Mu.L of the supernatant was subjected to SDS-PAGE, and the protein concentration of the kit was quantified using the protein.

(3) 40. Mu.L of 500mM DTT was added and incubated at 75℃for 30min; 80. Mu.L of 500mM IAA (protected from light) was incubated at room temperature for 30min.

(4) 100. Mu.L of 1% pancreatin acetic acid solution was added, overnight at 37 ℃.

(5) The next day, 3000g was centrifuged for 30 seconds, and the supernatant was incubated at 90℃for 10 minutes to terminate the enzymatic hydrolysis.

(6) Centrifuge 12000g for 30min, and collect 500. Mu.L of supernatant.

2.2 desalination

Desalting with Monospin C18 desalting column (GL Sciences Inc.), and obtaining desalted peptide.

2.3 suspending

The desalted peptide fragments were suspended by a vacuum concentrator. The dried peptide was resuspended in 500. Mu.L of 0.1% chromatographic pure formic acid before loading, and centrifuged at 12000g for 30min or passed through a PES filter of 0.22. Mu.m.

Example 3: qualitative and quantitative detection of 8 plant-derived allergens in processed food samples by isotope dilution mass spectrometry

A plurality of food samples are purchased from a supermarket, pretreatment of processed food samples is carried out according to the method of the embodiment 2, and a heavy standard peptide segment with the same concentration content as that of the standard curve drawing in the embodiment 1 is added into the supernatant obtained in the step 2.1, and then the steps of desalting and suspending are sequentially carried out. At least three replicates were run for each sample. Performing isotope dilution mass spectrometry detection on each sample by adopting the liquid chromatography condition and the mass spectrometry condition shown in the embodiment 1, and performing data processing by using the same parameters as those used in the drawing of a standard curve to obtain the area ratio of the light-standard peptide fragment to the heavy-standard peptide fragment; substituting the area ratio into a standard curve similar to the matrix, and calculating to obtain the concentration of the corresponding light-label peptide segment, which is also the concentration of the allergen protein.

The allergen content of the sample is calculated according to formula (1), and the calculated result retains two significant figures.

Wherein:

c, the content of the tested components in the test sample is expressed in milligrams per kilogram (mg/kg);

x-the concentration of the measured component solution obtained from the standard working curve in femtomoles per microliter (fmol/. Mu.L);

v-sample constant volume in milliliters (mL);

m-molecular weight of allergen protein in kilograms per mole (g/mol);

m-sample weight, unit gram (g).

With reference to the above method, various processed foods purchased were tested, and the test results are shown in table 7.

Table 7 quantitative results of allergen in food samples (n=3)

As can be seen from the above table, many foods contain more than one allergen, such as FA019 walnut black sesame paste samples, and contain soybean, hazelnut, walnut and sesame sensitization substances at the same time; the FA033 walnut cake contains the sensitization substances of wheat, soybean, walnut and sesame. The method can rapidly and accurately detect the types and the contents of various allergens contained in food samples, has strong specificity, high sensitivity and strong stability, and has good application effect in bread, biscuits, cakes, ice cream, yoghurt and the like.

The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.

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Claims (9)

1. A method for synchronously and quantitatively confirming multiple allergens in food based on mass spectrometry technology is characterized in that: detecting one or more items of the presence, the type and the absolute quantity of the allergen in the sample by utilizing an isotope dilution mass spectrometry according to the characteristic peptide fragment;

the allergen peptide comprises one or more of hazelnut characteristic peptide, walnut characteristic peptide, almond characteristic peptide, cashew characteristic peptide, wheat characteristic peptide, peanut characteristic peptide, soybean characteristic peptide, sesame characteristic peptide, milk characteristic peptide, egg characteristic peptide, fish characteristic peptide and shrimp characteristic peptide;

the characteristic peptide segment of each allergen protein comprises a light-standard peptide segment and a heavy-standard peptide segment, wherein the heavy-standard peptide segment is obtained by labeling one amino acid in the light-standard peptide segment by an isotope;

firstly, performing isotope dilution mass spectrometry detection on a light-standard peptide fragment and a heavy-standard peptide fragment with known concentrations to obtain a standard curve of the area ratio of the light-standard peptide fragment to the heavy-standard peptide fragment and the concentration ratio of the light-standard peptide fragment to the heavy-standard peptide fragment; and extracting peptide fragments in the food sample, adding the same-quantity heavy-standard peptide fragments for isotope dilution mass spectrometry detection, and comparing the detection result with a standard curve to realize the detection of the allergen in the sample.

2. The method for simultaneous quantitative determination of multiple allergens in a food based on mass spectrometry according to claim 1, wherein: the allergen peptide comprises at least two of hazelnut characteristic peptide, walnut characteristic peptide, almond characteristic peptide, cashew characteristic peptide, wheat characteristic peptide, peanut characteristic peptide, soybean characteristic peptide, sesame characteristic peptide, milk characteristic peptide, egg characteristic peptide, fish characteristic peptide and shrimp characteristic peptide;

preferably, the allergen peptide fragments comprise plant-derived hazelnut-characteristic peptide fragments, walnut-characteristic peptide fragments, almond-characteristic peptide fragments, cashew-characteristic peptide fragments, wheat-characteristic peptide fragments, peanut-characteristic peptide fragments, soybean-characteristic peptide fragments and sesame-characteristic peptide fragments; alternatively, the allergen peptide fragments include milk characteristic peptide fragments, egg characteristic peptide fragments, fish characteristic peptide fragments and shrimp characteristic peptide fragments of animal origin;

alternatively, the allergen peptide fragments include hazelnut, walnut, almond, cashew, wheat, peanut, soybean, sesame, milk, egg, fish, and shrimp peptide fragments.

3. The method for simultaneous quantitative determination of multiple allergens in a food based on mass spectrometry according to claim 2, wherein: the light-standard peptide and the heavy-standard peptide in the hazelnut characteristic peptide are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 or as SEQ ID NO.3 and SEQ ID NO. 4;

the light-standard peptide and the heavy-standard peptide in the walnut characteristic peptide are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6;

the light-standard peptide and the heavy-standard peptide in the almond characteristic peptide are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8;

the light-standard peptide and the heavy-standard peptide in the cashew nut characteristic peptide are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10;

the light-standard peptide and the heavy-standard peptide in the wheat characteristic peptide are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12;

the light-standard peptide and the heavy-standard peptide in the peanut characteristic peptide are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14;

the light-standard peptide and the heavy-standard peptide in the soybean characteristic peptide are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16;

the light-standard peptide and the heavy-standard peptide in the sesame characteristic peptide are respectively shown as SEQ ID NO.17 and SEQ ID NO. 18;

the light-standard peptide and the heavy-standard peptide in the milk characteristic peptide are respectively shown as SEQ ID NO.19 and SEQ ID NO.20 or as SEQ ID NO.21 and SEQ ID NO. 22;

the light-standard peptide and the heavy-standard peptide in the egg characteristic peptide are respectively shown as SEQ ID NO.23 and SEQ ID NO. 24;

the light-standard peptide and the heavy-standard peptide in the fish characteristic peptide are respectively shown as SEQ ID NO.25 and SEQ ID NO. 26; the light-standard peptide and the heavy-standard peptide in the shrimp characteristic peptide are respectively shown as SEQ ID NO.27 and SEQ ID NO. 28;

SEQ ID NO.1 ADIYTEQVGR

SEQ ID NO.2 ADIYTEQV*(13C5,15N)GR

SEQ ID NO.3 TNDNAQISPLAGR

SEQ ID NO.4 TNDNAQISPL*(13C6,15N)AGR

SEQ ID NO.5 ISTVNSHTLPVLR

SEQ ID NO.6 ISTVNSHTLPVL*(13C6,15N)R

SEQ ID NO.7 GNLDFVQPPR

SEQ ID NO.8 GNLDFV*(13C5,15N)QPPR

SEQ ID NO.9 ADIYTPEVGR

SEQ ID NO.10 ADIYTPEV*(13C5,15N)GR

SEQ ID NO.11 YFIALPVPSQPVDPR

SEQ ID NO.12 YFIALPVPSQPV*(13C5,15N)DPR

SEQ ID NO.13 RPFYSNAPQEIFIQQGR

SEQ ID NO.14 RPFYSNAPQEIFIQQGR*(13C6,15N4)

SEQ ID NO.15 VLIVPQNFVVAAR

SEQ ID NO.16 VLIVPQNFVV*(13C5,15N)AAR

SEQ ID NO.17 AFYLAGGVPR

SEQ ID NO.18 AFYLAGGV*(13C5,15N)PR

SEQ ID NO.19 HQGLPQEVLNENLLR

SEQ ID NO.20 HQGLPQEVLNENLL*(13C6,15N)R

SEQ ID NO.21 TPEVDDEALEK

SEQ ID NO.22 TPEVDDEAL*(13C6,15N)EK

SEQ ID NO.23 GGLEPINFQTAADQAR

SEQ ID NO.24 GGLEPINFQTAADQAR*(13C6,15N4)

SEQ ID NO.25 TIDDLEDELYAQK

SEQ ID NO.26 TIDDLEDELYAQK*(13C6,15N2)

SEQ ID NO.27 ALSNAEGEVAALNR

SEQ ID NO.28 ALSNAEGEVAAL*(13C6,15N)NR。

4. the method for simultaneous quantitative determination of multiple allergens in a food based on mass spectrometry according to claim 1, wherein: the liquid chromatography conditions were: mobile phase a was 0.1% formic acid/water, mobile phase B was 0.1% formic acid/acetonitrile, flow rate 0.2mL/min; column temperature 40 ℃; the chromatographic column is a C18 chromatographic column; the liquid chromatographic gradients are shown in the following table;

the mass spectrum conditions are as follows: using HESI source positive ion reaction mode, PRM scanning mode; the ion source parameters of the atomizing gas, the gas curtain gas, the auxiliary heating gas and the collision gas are high-purity nitrogen; PRM scan set resolution of 70,000, C-trap maximum capacity of 1X 10 ⁶ The maximum injection time of the C-trap is 100ms, the isolation window is 1.6m/z, and the fragmentation collision energy of the peptide fragment is set to be 27;

the inclusion list is set up as follows;

5. use of the method according to any one of claims 1-4 for simultaneous quantitative determination of a plurality of allergens in a food based on mass spectrometry technology for detection of processed food allergens.

6. The use according to claim 5, characterized in that: diluting a storage solution containing the light-standard peptide fragment with a food matrix solution without the light-standard peptide fragment, adding the heavy-standard peptide fragment, detecting by using an isotope dilution mass spectrometry, and drawing to obtain a standard curve; pretreating a processed food sample to be detected, adding an equivalent weight-marked peptide fragment, performing isotope dilution mass spectrometry detection, and judging whether the processed food sample to be detected contains a corresponding light-marked peptide fragment according to the existence of a characteristic peak;

and/or comparing the characteristic peak area detection result with a standard curve to calculate the concentration of the corresponding light-label peptide segment.

7. The use according to claim 6, characterized in that: the quantitative standard curves of the characteristic peptide fragments in different matrixes are shown in the following table;

wherein Y is the ion peak area ratio of the light-standard peptide fragment/heavy-standard peptide fragment of the tested component, and X is the concentration of the tested component.

8. The use according to claim 7, characterized in that: calculating the content of the allergic substances by the formula 1;

wherein:

c, the content of the tested components in the test sample is expressed in milligrams per kilogram (mg/kg);

x-the concentration of the measured component solution obtained from the standard working curve in femtomoles per microliter (fmol/. Mu.L);

v-sample constant volume in milliliters (mL);

m-molecular weight of allergen protein in kilograms per mole (g/mol);

m-sample weight, unit gram (g).

9. The use according to claim 6, characterized in that: the pretreatment process of the processed food sample to be detected sequentially comprises the processes of protein extraction, reduction, alkylation, enzymolysis, desalination and suspension;

the preparation of the food matrix solution sequentially comprises the processes of protein extraction, reduction, alkylation, enzymolysis, desalination and suspension drying.

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