CN111089910B - Method for assisting in identifying linear epitope of plant food allergen - Google Patents
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Abstract
The invention provides a method for assisting in identifying linear epitopes of plant food allergens, which comprises the following steps: taking a product of vegetable food protein after simulated gastrointestinal digestion as a sample, and separating by adopting high performance liquid chromatography; carrying out mass spectrum detection on the separated sample to obtain mass spectrum information of the digestion-resistant peptide fragment; and then comparing and analyzing with the whole amino acid sequence of the plant food allergen to obtain a peptide segment matched with the plant food allergen. The peptide fragments obtained by the method have correlation with linear epitopes of the allergen, or are covered or crossed. Therefore, the range of identifying the linear epitope can be reduced, the method is more targeted, and compared with the existing method for identifying the linear epitope, the auxiliary method reduces the workload of the whole identification process and ensures that the identification result is more accurate.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to a method for assisting in identifying linear epitopes of plant food allergens.
Background
Food allergy is an adverse reaction that occurs when the body is exposed to a specific food, i.e. an allergic reaction that can involve different organs of the body due to the specific immune response of the immune system to allergens. According to epidemiological investigation, the incidence rate of infants and young children worldwide reaches 8 percent, and the incidence rate of adults reaches 5 percent. Food allergy is mostly type I hypersensitivity mediated by IgE, can cause systemic multi-system reactions such as asthma, diarrhea, urticaria, allergic dermatitis and the like, even can generate shock to endanger life, and seriously affects the life quality of patients.
Food allergens in nature are of various types and mainly originate from plants, animals and microorganisms, but most of the allergens causing food allergic diseases originate from plants. To date, the world health organization/international association of immunology (WHO/IUIS) allergen nomenclature group committee has identified a total of 314 food allergens, 216 of which are of plant origin. Four of eight types of allergic foods reported by the Food and Agriculture Organization (FAO) of the United nations are vegetable foods, namely peanuts, soybeans, nuts and wheat.
Compared with various proteins existing in the biological world, the vegetable food allergen protein only accounts for a small part, and the conserved structure and biological activity of the protein determine the characteristics of sensitization. The majority of plant food allergens were found to belong to the prolamin superfamily, Cupin superfamily, arrestin superfamily and Bet v 1 related protein family, classified according to the similar amino acid sequences and three-dimensional structures possessed by proteins. Most allergens of the Cupin superfamily belong to the family of seed storage proteins, including legume, tree nut and seed allergens. The prolamin superfamily is the most prominent seed storage protein of cereals (except rice and oats) and is rich in proline and glutamine. Hypersensitive inhibitory proteins are mainly found in flowering plants, such as plant pollen, fruits and vegetables. The Bet v 1-related protein has a similar three-dimensional structure in space, most of which contain 7 β sheets and 3 α -helical structures, and a hydrophilic helical structure at the C-terminus.
The surface of food allergen molecule has specific structure and amino acid sequence, and can stimulate body to produce immune response, and the immune active region is called antigen epitope. According to the spatial distribution characteristics of amino acids in the epitope, the epitope can be divided into continuous epitope and discontinuous epitope. The continuous epitope is also called linear epitope and is continuously composed of amino acids in a primary sequence; discontinuous epitopes, also known as conformational epitopes, are not contiguous in the primary sequence, but can be brought into close proximity to one another on the surface of a properly folded protein spatial structure, forming an epitope that is specifically recognized by the antibody. Studies have shown that more than 90% of all epitopes belong to conformational epitopes, and less than 10% are linear epitopes. However, food allergens are digested by the stomach and small intestine after being taken orally, factors such as gastric acid, digestive enzymes and the like can destroy a large number of conformational epitopes, linear epitopes can still be identified, and the organism is stimulated to generate anaphylactic reaction. Therefore, the study of linear epitopes of food allergens is of great importance for the prevention and treatment of food allergy.
At present, methods for identifying antigen linear epitopes mainly include polypeptide synthesis technology, amino acid site-directed mutagenesis technology and the like. The polypeptide synthesis technology is characterized in that a synthesized short overlapping peptide fragment is incubated with serum of an allergic patient through a dot blot or immunoblot experiment, and the binding condition of the short overlapping peptide fragment and the serum of a specific IgE antibody is detected, so that a linear epitope is judged and screened, but the serum of the allergic patient needs to be collected, the time consumption is long, the workload is large, the blindness is high, the epitope in an overlapping region can be ignored, and the accuracy is difficult to guarantee. The amino acid site-directed mutagenesis technology is to screen out a main epitope by sequentially mutating one or more specific amino acids of a target protein and then comparing the binding degree of a natural target protein and a mutated recombinant protein with an antibody, and the whole process has large mutation, recombination and screening workload.
Therefore, a method for assisting in identifying linear epitopes of plant food allergens is needed, so that the identification workload is reduced, and the result is more accurate.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for assisting in identifying the linear epitope of the plant food allergen, so that the identification workload is reduced, and the result is more accurate.
The invention provides a method for assisting in identifying linear epitopes of plant food allergens, which comprises the following steps:
(1) taking a product of vegetable food protein after simulated gastrointestinal digestion as a sample, and separating by adopting high performance liquid chromatography;
(2) performing mass spectrum detection on the sample separated in the step (1) to obtain mass spectrum information of the digestion-resistant peptide fragment;
(3) and (3) comparing and analyzing the mass spectrum information of the digestion-resistant peptide fragment obtained in the step (2) with the whole amino acid sequence of the plant food allergen to obtain the peptide fragment matched with the plant food allergen.
In the technical scheme, the mass spectrum information of the anti-digestion peptide fragment is detected based on high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), and then is compared and analyzed with the whole amino acid sequence of the allergen to obtain the peptide fragment matched with the allergen, and the peptide fragments have correlation with the linear epitope of the allergen or are covered or crossed, so that the range of identifying the linear epitope can be reduced to the anti-digestion peptide fragment matched with the allergen through the technical scheme, and then the linear epitope of the allergen is further identified by combining other means such as bioinformatics and the like. Compared with the existing method for identifying the linear epitope, the auxiliary method reduces the workload of the whole identification process and ensures that the identification result is more accurate.
Preferably, said simulated gastrointestinal digestion in step (1) is by pepsin or pancreatin digestion.
Preferably, the digestion time is 50-70 min, and more preferably 60 min.
Preferably, when the high performance liquid chromatography is adopted for separation in the step (1), the buffer solution A is 0.05-0.2% formic acid aqueous solution, and the buffer solution B is 0.05-0.2% formic acid acetonitrile solution.
The buffer solution A and the buffer solution B are adopted, so that the stability of a sample in the detection process is facilitated, and the proper separation degree and sensitivity are obtained.
Preferably, the buffer solution a is a 0.1% formic acid aqueous solution, and the buffer solution B is a 0.1% formic acid acetonitrile solution.
Preferably, the chromatographic column is equilibrated with 95-98% of said buffer solution A; the flow rate of the sample is 500-700 nL/min.
Preferably, when mass spectrometry is performed in the step (2), the scanning range is 300-1400 m/z.
As a preferred embodiment, the method for assisting in identifying linear epitopes of plant food allergens comprises the following steps:
(1) taking a product of vegetable food protein digested by pepsin or pancreatin for 50-70 min as a sample, and separating by adopting a high performance liquid chromatography, wherein the chromatographic separation conditions are as follows: the buffer solution A is 0.05-0.2% formic acid aqueous solution, the buffer solution B is 0.05-0.2% formic acid acetonitrile solution, the chromatographic column is balanced by 95-98% of the buffer solution A, and the flow rate of the sample is 500-700 nL/min;
(2) performing mass spectrum detection on the sample separated in the step (1), wherein the scanning range of a mass spectrometer is 300-1400 m/z, and obtaining mass spectrum information of the digestion-resistant peptide fragment;
(3) and (3) comparing and analyzing the mass spectrum information of the digestion-resistant peptide fragment obtained in the step (2) with the whole amino acid sequence of the plant food allergen to obtain the peptide fragment matched with the plant food allergen.
The invention detects out the mass spectrum information of the anti-digestion peptide section based on the high performance liquid chromatography tandem mass spectrum, and then carries out the comparative analysis with the amino acid complete sequence of the allergen to obtain the peptide section matched with the allergen, and the peptide sections have the correlation with the linear epitope of the allergen or are covered or crossed. Therefore, the method can reduce the range of identifying the linear epitope, is more targeted, and reduces the workload of the whole identification process and ensures more accurate identification result compared with the existing method for identifying the linear epitope.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a primary mass spectrum of a pepsin digestion product of peanut protein in example 1 of the present invention;
FIG. 2 is a primary mass spectrum of a product of the trypsinization of peanut proteins in example 1 of the present invention;
FIG. 3 shows the positioning of the digestion-resistant peptide fragment in the amino acid sequence of Jug r 2 in example 2 of the present invention;
FIG. 4 shows the amino acid composition of Jug r 2 digestion-resistant peptide fragment in example 2 of the present invention;
FIG. 5 shows the positioning of the digestion-resistant peptide fragment and the predicted epitope on the amino acid sequence of CM16 in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The peanut allergen Ara h1 is 7S globulin, belongs to Cupin superfamily, and can be identified by more than 90% of peanut allergy patient serum through serological analysis. The structural sequence of Cupin superfamily has the characteristics of high conservation, strong heat stability, enzymolysis resistance and indigestibility, so Ara h1 is selected as the model allergen in the invention.
The embodiment provides a method for assisting in identifying linear epitopes of peanut allergen Ara h1, which comprises the following steps:
s1, taking a product of peanut protein after being digested for 60min by pepsin as a sample, and separating by adopting a nanoliter HPLC liquid phase system Easy-nLC1000, wherein a buffer solution: the solution A is 0.1% formic acid water solution, and the solution B is 0.1% formic acid acetonitrile solution; the chromatographic column is balanced by 95 percent of solution A; the sample is loaded to a pre-column C18trap column (3mm, 0.10X 20mm) by an autosampler, and then separated by an analytical column C18column (1.9mm, 0.15X 120mm) at a flow rate of 600 nL/min; the relevant liquid phase gradients are shown in table 1:
TABLE 1 liquid chromatography elution gradient parameters
S2, detecting the separated sample by a Q-exact mass spectrometer, wherein the main parameters of the mass spectrometer comprise: the scanning type is Full MS, the scanning range is 300-1400 m/z, polar selects positive ion scanning, the resolution is 70000, Microscan selects 1, AGC target selects 3e6Max injection time is selected to be 60 ms. The primary mass spectrum of the sample obtained by detection is shown in figure 1, wherein each spectrum peak represents one kindPeptide fragments, the composition of which can be determined by secondary mass spectrometry, 5302 peptide fragments are identified by primary mass spectrometry, and the mass-to-charge ratio (m/z) of the polypeptide mixture is intensively distributed between 300-500;
s3, acquiring the peanut allergen protein sequence from the Uniprot database for pFind search engine retrieval, wherein the retrieval parameters are set as follows: the enzyme is protease or pancreatin; missed cutting sites: 2; fixing and modifying: an acyl methylation modification (C); variable modification: acetylation (N-terminal), glutamine → pyroglutamic acid (N-terminal), oxidation (M); precursor mass deviation: 1.5 Da; fragment mass deviation: 0.5 Da; peptide fragment reliability: high; length of peptide fragment: > 4; peptide fragment FDR: less than or equal to 0.01.
As a result, 28 peptides belonging to the peanut allergen protein were searched out in total, and 15 of them were matched with Ara h1 and shown in Table 2. The 15 polypeptides covered 19.38% of the full Ara h1 sequence and were predominantly distributed at the amino terminus of the protein. Wherein, cleavage at 299-313 yielded three fragments, and cleavage at 401-411 and 415-426 yielded two fragments, respectively, indicating that these regions have digestion stability. Compared with the linear epitope of the known Ara h1, the polypeptides No. 5-15 are all crossed with the linear epitope by partial amino acid sequences, and the polypeptides No. 5-7 and 9-12 cover 2 linear epitope sequences.
TABLE 2 Pepsin digestion resistant peptide fragment and Ara h1 matched peptide fragment
Taking the product of peanut protein digested by trypsin for 60min as a sample, repeating the steps of S1-S3, and obtaining a primary mass spectrum of the sample through detection as shown in figure 2, wherein 5037 peptide fragments are identified in total, the mass-to-charge ratio distribution of the peptide fragments is similar to that of gastric enzyme digestion, and the mass-to-charge ratio distribution is also concentrated at 300-500 m/z. A total of 8 peptides belonging to peanut allergen protein were detected by pFind search engine, of which 4 matched Ara h1, as shown in Table 3, covering 6.39% of the entire sequence of Ara h 1. The anti-digestibility of the epitope is proved by comparing with the known linear epitope of Ara h1, wherein the peptide fragments No. 1 and No. 2 cover 94-104 sites, and the known linear epitope exists near the amino acid sequence of the other 2 peptide fragments.
TABLE 3 pancreatic digestion resistant peptide fragment and Ara h1 matched peptide fragment
As a result, the digestion-resistant peptide fragment of Ara h1 contains a known linear epitope near the position of the complete protein sequence, and there are some digestion-resistant overlapping peptide fragments corresponding to the epitope; more peptide fragment information can be obtained after digestion by gastric enzyme than that by pancreatic enzyme, which is helpful for identifying more linear epitopes. In short, the identification range can be narrowed to the anti-digestion peptide matched with the allergen by the auxiliary identification method of the embodiment, and the linear epitope of the allergen can be further identified by combining other means such as bioinformatics and the like.
Example 2
The walnut allergen Jug r 2 belongs to the family of 7S piscine, is in the same family as the peanut allergen Ara h1, has a primary sequence consisting of 593 amino acids, has a molecular weight of about 44kDa, and exists as a trimer oligomer under natural conditions.
The embodiment provides a method for assisting in identifying walnut allergen Jug r 2 linear epitope, which comprises the following steps:
s1, taking a product of the walnut protein after being digested for 60min by pepsin as a sample, and separating by adopting a nano-liter HPLC liquid phase system Easy-nLC1000, wherein a buffer solution: the solution A is 0.1% formic acid water solution, and the solution B is 0.1% formic acid acetonitrile solution; the chromatographic column is balanced by 95 percent of solution A; the sample is loaded to a pre-column C18trap column (3mm, 0.10X 20mm) by an autosampler, and then separated by an analytical column C18column (1.9mm, 0.15X 120mm) at a flow rate of 600 nL/min;
s2, detecting the separated sample by a Q-exact mass spectrometer, and detecting 4953 peptide fragments;
s3, obtaining a walnut allergen protein sequence from a Uniprot database, and using the walnut allergen protein sequence for retrieval by a pFind search engine, wherein the retrieval parameters are set as follows: the enzyme is protease or pancreatin; missed cutting sites: 2; fixing and modifying: an acyl methylation modification (C); variable modification: acetylation (N-terminal), glutamine → pyroglutamic acid (N-terminal), oxidation (M); precursor mass deviation: 1.5 Da; fragment mass deviation: 0.5 Da; peptide fragment reliability: high; length of peptide fragment: > 4; peptide fragment FDR: less than or equal to 0.01.
The results found 11 peptides belonging to the walnut allergen protein, of which 4 matched Jug r 2 covering 11% of the full amino acid sequence, are listed in Table 4.
TABLE 4 Pepsin digestion resistant peptide fragment and Jug r 2 matched peptide fragment
Taking the product of the walnut protein after being digested by pancreatin for 60min as a sample, repeating the steps S1-S3, detecting 6493 peptide segments in total, and detecting 44 peptide segments belonging to peanut allergen protein in total by using a pFind search engine, wherein 13 peptide segments are matched with Jug r 2 and listed in Table 5, and cover 46% of the amino acid full sequence of Jug r 2.
TABLE 5 pancreatic digestion resistant peptide fragments matched with Jug r 2
To further observe the distribution of these digestion-resistant peptide fragments over the primary amino acid sequence of allergen Jug r 2, the sequence of Jug r 2 was obtained from the NCBI database and the peptide fragments were labeled as shown in FIG. 3, in which the digestion-resistant peptide fragments are boxed. Taken together, these digestion-resistant peptide fragments were predominantly distributed in the C-terminal region of Jug r 2, covering about 74% of the sequence at positions 428-572. Wherein pepsin digestion produced two overlapping peptide fragments at 515-528, and tryptic digestion produced overlapping peptide fragments at 470-483, 488-513, and 545-572, respectively, demonstrating the anti-digestibility of these regions. In addition, the digestion-resistant peptides No. 6-13 under the action of trypsin are almost linked to each other, covering the entire sequence at the 470-572 site. It is noteworthy that there is a region of overlap between the pepstatin-resistant peptides No. 2-4 and the pancreatin-resistant peptides No. 8-11, and that the high digestive resistance of this region is likely to retain a large number of linear epitopes.
The frequency of occurrence of various amino acids in the Jug r 2 digestion-resistant peptide fragment was analyzed using Bioedit software, and the results are shown in FIG. 4. The Jug r 2 digestion resistant peptide fragment consists of 19 amino acids and lacks cysteine (Cys) residues constituting the complete sequence. In these peptide stretches, which are rich in negatively charged glutamic acid (Glu) and positively charged arginine (Arg), hydrophobic amino acids, compared to the amino acid composition of the full sequence, such as: the frequency of alanine (Ala), leucine (Leu), isoleucine (Ile), and the like increases. The amino acids in the peptide fragments are classified and counted, wherein the hydrophilic amino acid accounts for 43 percent, the neutral amino acid accounts for 15 percent, and the hydrophobic amino acid accounts for 42 percent. The result shows that the amino acid composition of the Jug r 2 digestion-resistant peptide segment has similarity with the amino acid composition of the Cupin superfamily pattern antigen Ara h1 linear epitope, and the region may contain unknown epitope.
And identifying the digestion-resistant peptide fragments of the Jug r 2 by using high performance liquid chromatography/mass spectrometry, determining 11 non-overlapping amino acid sequences, and mainly distributing in a C-terminal region. Through amino acid composition and spatial structure positioning analysis, the other 10 peptide segments are found to accord with the composition characteristics of the linear epitope in the Cupin superfamily and possibly contain the linear epitope except that the sequence at the 215-220 site is located in the trimer core and does not have solvent accessibility. Comparing the digestion-resistant peptide fragment of the Jug r 2 with the linear epitope of Ara h1, a plurality of overlapping regions exist between the two, namely the Jug r 2 digestion-resistant peptide fragment can cause the Ara h1 allergic patients to generate cross-allergic reaction.
In conclusion, the digestion-resistant peptide fragment of Jug r 2 has the same distribution rule as the linear epitope of the Cupin superfamily pattern antigen Arah 1, and may contain unknown linear epitope, so the identification range can be narrowed to the obtained Jug r 2 digestion-resistant peptide fragment by the auxiliary identification method of the embodiment, and then the specific linear epitope is further identified.
Example 3
The wheat allergen CM16 is a protein with a molecular weight of 17kDa and composed of 143 amino acids, belongs to prolamin superfamily, contains a plurality of cysteine residues, can form intramolecular disulfide bond, ensures the structural stability of the protein, and has heat resistance.
The embodiment provides a method for assisting in identifying a linear epitope of wheat allergen CM16, which comprises the following steps:
using the product of wheat protein digested with trypsin for 60min as a sample, adopting the steps of S1-S3 in the same example 1 to detect 6753 peptide fragments, constructing a search database according to the amino acid sequence of wheat allergen provided by the Uniprot database, and detecting 23 peptide fragments belonging to wheat allergen protein by using a pFind search engine, wherein the majority of the peptide fragments are derived from an alpha-amylase/trypsin inhibitor subtype and a high molecular weight glutelin subunit, and 3 of the peptide fragments are matched with CM16, as shown in table 6.
TABLE 6 pancreatic digestive peptide fragments matching wheat major allergens
Wherein the high molecular weight glutelin (HMW-GS) belongs to prolamin superfamily, generally contains 630-830 amino acids, and consists of hydrophilic regions at N end and C end and 3 regions of middle hydrophobic repetitive structural domain. As HMW-GS has larger molecular weight, the structure has repeatability and does not have a modeling template with higher homology, and accurate and deep analysis cannot be carried out, the alpha-amylase/trypsin inhibitor CM16 is selected subsequently to carry out the research on the relation between the linear epitope and the digestion-resistant peptide segment.
The possible linear epitope for CM16 was predicted using DNAStar software and the results are shown in table 7.
TABLE 7DNAStar predicts the resulting linear epitope of CM16
The distribution of the predicted epitope and the anti-tryptic peptide fragment over the primary amino acid sequence of allergen CM16 is labeled as shown in fig. 5, with the box representing the predicted linear epitope and the underlined representing the anti-tryptic peptide fragment. In the whole view, the predicted epitope and the digestion-resistant peptide fragment are mainly distributed in the middle and the C terminal, and the digestion-resistant peptide fragments No. 1 and No. 2 and the predicted epitope No. 2 have partial sequence coincidence, which shows that the linear epitope has digestion resistance, and simultaneously supports the feasibility and the applicability of the method for assisting in identifying the linear epitope.
By combining the results, the invention establishes a method for detecting the peptide fragment sequence of the plant food allergen by utilizing the high performance liquid chromatography/mass spectrometry and identifying the linear epitope of the allergen in an auxiliary way, is suitable for the allergens of the Cupin superfamily and the prolamin superfamily, and is suitable for the plant food allergens of other families. Compared with the prior art, the method is more targeted and more accurate, reduces the workload, can further identify the linear epitope by combining bioinformatics or other means after the preliminary identification of the anti-digestion peptide segment, and has important significance for developing a novel and accurate method for detecting the linear epitope. The method is suitable for identifying all food allergen linear epitopes which have complete spatial structures and are easy to digest in the gastrointestinal tract.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (1)
1. A method for assisting in identifying a linear epitope of wheat allergen CM16, comprising the following steps:
s1, taking a product of wheat protein after being subjected to trypsinization for 60min as a sample, and separating by adopting a nanoliter-grade HPLC liquid phase system Easy-nLC1000, wherein a buffer solution: the solution A is 0.1% formic acid water solution, and the solution B is 0.1% formic acid acetonitrile solution; the chromatographic column is balanced by 95 percent of solution A; the sample is loaded to a pre-column C18trap column with the diameter of 3 mu m and the diameter of 0.10 multiplied by 20mm by an automatic sample injector, and then is separated by an analytical column C18column with the diameter of 1.9 mu m and the diameter of 0.15 multiplied by 120mm, and the flow rate is 600 nL/min; the liquid chromatography elution gradient parameters were as follows:
s2, detecting the separated sample by a Q-exact mass spectrometer, wherein the main parameters of the mass spectrometer comprise: the scanning type is Full MS, the scanning range is 300-1400 m/z, polar selects positive ion scanning, the resolution is 70000, Microscan selects 1, AGC target selects 3e6Max injection time is selected for 60ms, and 6753 peptide fragments are detected;
s3, constructing a retrieval database according to the amino acid sequence of the wheat allergen provided by the Uniprot database, and detecting 23 peptide fragments belonging to the wheat allergen protein by using a pFind search engine, wherein 3 peptide fragments are matched with CM16, and the table is as follows:
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