A method for detecting sesame allergen proteins in processed foods

Abstract

This invention relates to a mass spectrometry method for detecting seven sesame allergenic proteins in processed foods. The method uses 12 sesame processed products (roasted, ground, boiled, baked, fried, and steamed) as samples. Protein extraction and trypsin digestion are performed, and the hydrolyzed peptides are analyzed by high-resolution mass spectrometry. Characteristic peptides of sesame allergenic proteins are initially screened based on principles such as response intensity and the number of amino acids (6-15). The specificity of these peptides is analyzed using BLAST, and specificity verification is performed to identify nine characteristic peptides with processing stability. Mass spectrometry parameters are optimized for each ion pair of peptides, and a multiple reaction monitoring (MRM) method is established. This method can sensitively and with high throughput detect sesame allergens in processed foods, which not only helps to standardize allergen labeling but also helps to protect the health of allergy sufferers, and is of great significance.

Description

Technical Field

[0001] This invention relates to a method for detecting sesame allergenic proteins in processed foods. Specifically, it relates to a method for detecting seven sesame allergenic proteins in processed foods using characteristic peptides with processing stability. Background Technology

[0002] Sesame (Sesamumindicum L.), belonging to the genus Sesamum in the family Pedaliaceae, is a traditional oilseed crop, mainly cultivated in Henan, Hubei, Anhui, and Jiangxi provinces. my country is a major producer and consumer of sesame. Sesame seed coats come in various colors, exhibiting a continuous variation from white to black, including white, brown, and black. Different colored sesame seeds have different applications in the food industry. White sesame seeds are mostly used as a raw material for food products, such as bread and biscuits; yellow and other variegated sesame seeds are mostly used to prepare sauces or for oil extraction; black sesame seeds are mainly used to produce sesame paste and sesame powder. Due to its anti-aging, antioxidant, and immune-boosting properties, sesame is also used as a functional ingredient in health foods and novel food products, making it widely used in the food industry.

[0003] Sesame seeds are a common food allergen. The World Health Organization / International Union of Immunological Societies (WHO / IUIS) has identified seven main sesame allergen proteins (Ses i 1-Ses i 7). Countries such as the EU, Australia and New Zealand, Canada, and the United States require the labeling of sesame allergen information on food labels. However, during production, storage, and transportation, due to the shared use of production lines or equipment, sesame seeds may be mixed into unlabeled foods, affecting the health of allergy sufferers. Furthermore, food allergen labeling is not standardized, often using vague terms such as "may contain" for identification.

[0004] Currently, the detection of sesame allergens mainly focuses on the nucleic acid and protein levels. Nucleic acid-based methods include molecular biology techniques such as polymerase chain reaction (PCR), while protein-based methods include immunological methods and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). Due to the complexity of processed food matrices and the impact of processing techniques on protein structure and nucleic acid integrity, the application of molecular biology and immunological methods in the detection of sesame allergens in processed foods is limited. By identifying characteristic peptides of target allergen proteins, HPLC-MS can simultaneously detect multiple allergen proteins, achieving high-sensitivity detection. Previous studies have reported the screening of three characteristic peptides of the sesame allergen protein Sesi 6 for the detection of sesame allergen proteins in bread and biscuits. Another study identified characteristic peptides of seven sesame allergen proteins and used the isotope internal standard method to quantify these seven sesame allergen proteins in food. In all the above studies, raw sesame seeds were used as samples for the screening of characteristic peptides.

[0005] However, sesame seeds require processing to develop their aroma and be used in food. Seven sesame allergen proteins exhibit different characteristics, and their structures are affected by processing techniques. Roasting white sesame seeds at 150℃ for 15 minutes alters the secondary structure of sesame allergen proteins, increasing random coils and decreasing antiparallel β-sheets. When sesame seeds are treated with lower pressures (100 MPa, 200 MPa), random coils, α-helices, and β-sheets transform, resulting in a more ordered protein structure. Using sesame seeds as a sample, 12 characteristic peptides of sesame proteins were screened, but only 8 of these were identified in cold-pressed vegetable oil. This indicates that high-pressure processing does indeed affect the structure of sesame allergen proteins, thus impacting the accuracy of detection. Therefore, it is necessary to screen for characteristic peptides with processing stability to facilitate the detection of sesame allergen proteins in processed foods and improve detection accuracy. Summary of the Invention

[0006] The purpose of this invention is to identify nine characteristic peptides with good specificity and processing stability using sesame products prepared by different processing techniques as samples, thereby providing a method for detecting seven sesame allergen proteins in processed foods. This method achieves high-throughput and high-sensitivity detection of sesame allergens in processed foods, resulting in accurate and stable detection results.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] A method for detecting seven sesame allergenic proteins in processed foods includes the following steps:

[0009] (1) Extraction of crude protein from sesame: The sesame sample was defatted, and a protein extraction buffer solution was added. The crude protein extract was obtained by oscillation combined with water bath ultrasound.

[0010] (2) Trypsin digestion: The crude sesame protein extract obtained in step (1) was digested with trypsin to obtain hydrolyzed peptides;

[0011] (3) Analysis of hydrolyzed peptides based on ultra-high performance liquid chromatography-time-of-flight high-resolution mass spectrometry: Determining chromatographic and mass spectrometric conditions for analyzing hydrolyzed peptides;

[0012] (4) Screening of characteristic peptides: Hydrolyzed peptides were identified by targeted analysis using ProteinPilot software, and 9 characteristic peptides with processing stability were identified from 7 sesame allergen proteins after specificity verification.

[0013] (5) Establishment of Scheduled MRM method: Mass spectrometry parameters were optimized for 9 characteristic peptide ion pairs of sesame allergens. A Scheduled MRM method was established based on high performance liquid chromatography-triple quadrupole mass spectrometry to detect 7 sesame allergen proteins in processed foods.

[0014] The detection method of the present invention is further preferred as follows:

[0015] (1) Crude protein extraction from sesame: A suitable amount of sesame sample was cryogenically ground in liquid nitrogen. Hexane was added at a ratio of sesame:hexane = 1:5, stirred at room temperature for 20 min, centrifuged (4000 rpm, 10 min), and the supernatant was discarded. The defatting process was repeated three times. 1 g of defatted sesame powder was weighed, and 10 mL of protein extraction buffer solution (50 mM Tris-HCl + 7 M urea + 2 M thiourea + 4% CHAPS, pH 8.5) was added. The mixture was vortexed for 1 min, and extracted by shaking at room temperature for 20 min. The mixture was then sonicated in a water bath for 20 min. After centrifugation (12000 rpm, 20 min), the supernatant was filtered through a 0.22 μm filter membrane, and the protein concentration of the crude extract was determined.

[0016] (2) Trypsin digestion: Take about 200 μg of protein into a centrifuge tube and add water to a total volume of 50 μL. Add 10 μL of 120 mM DTT solution and incubate in a water bath (37℃, 1 h). Add 10 μL of 600 mM IAA solution and react in the dark for 15 min. Transfer the liquid to a 5 kDa ultrafiltration membrane, centrifuge (12000 rpm, 20 min), add 100 μL of 50 mM ABC solution, centrifuge (12000 rpm, 15 min), and repeat three times. Continue to add 100 μL of 50 mM ABC solution, add 4 μL of 1 μg / μL trypsin at a 1:50 mass ratio, mix well, and incubate in a water bath at 37℃ for 10 h. Remove the ultrafiltration tube, centrifuge (12000 rpm, 20 min), add 100 μL of 25 mM ABC solution, centrifuge (12000 rpm, 15 min), and repeat three times. Place the ultrafiltration tube in a vacuum rotary evaporator and dry by rotation. Add 100-200 μL of mass spectrometer water and continue drying by rotation. Repeat 3 times. Add 100 μL of mobile phase A (98% water-2% acetonitrile-0.1% formic acid), vortex to mix, centrifuge (10000 rpm, 10 min), and transfer 80 μL of the liquid to a liquid chromatography vial. Store at 4°C for a short period.

[0017] (3) Hydrolyzed peptides were analyzed using ultra-high performance liquid chromatography-time-of-flight high-resolution mass spectrometry, under the following conditions:

[0018] Chromatographic conditions: The chromatographic column was an Xbridge Peptide BEH C18 (4.6 mm × 150 mm, 3.5 μm). Waters Corporation); Mobile phase A: 98% water - 2% acetonitrile - 0.1% formic acid; Mobile phase B: 98% acetonitrile - 2% water - 0.1% formic acid; Flow rate: 0.25 mL / min; Column temperature: 40 °C; Injection volume: 5 μL; Gradient elution program: 0–0.5 min, 5%–8% B; 0.5–0.6 min, 8%–12% B; 0.6–25 min, 12%–30% B; 25–30 min, 30%–35% B; 30–30.5 min, 35%–80% B; 30.5–38 min, 80% B; 38–38.5 min, 80%–5% B; 38.5–50 min, 5% B.

[0019] Mass spectrometry conditions: Nanospray III ion source; ESI+ positive ion mode; spray voltage 2500V; nebulizer gas 6psi; curtain gas 30psi; temperature 150℃; information-dependent acquisition mode; TOF MS mode 350~1500m / z, 250ms; TOFMS / MS mode 100~1500m / z, 100ms, precursor ion charge range +2~+5; dynamic exclusion time 20s; cycle time 2s; Rolling CE enabled, runtime 50min.

[0020] (4) Screening of characteristic peptides: Hydrolyzed peptides were identified by targeted analysis using ProteinPilot software. Peptides with high response intensity, 6-15 amino acids, no missed cleavage sites, and no non-enzymatic cleavage were selected as pre-selected characteristic peptides. The specificity of these peptides was analyzed using BLAST, and the specificity was verified using different varieties of sesame (Table 3) and common food allergens (Table 4) as samples.

[0021] Table 1. Seven main allergenic proteins in sesame seeds.

[0022]

[0023] Table 27 Characteristic Peptides of Sesame Allergen Proteins

[0024]

[0025] Table 3 Information on positive samples for specificity verification

[0026]

[0027] Table 4 Information on negative samples for specificity verification

[0028]

[0029] (5) Establishment of Scheduled MRM Method: Characteristic peptide ion pair information was obtained using the open-source software Skyline. Mass spectrometry parameters were optimized based on theoretical values ​​of declustering voltage and collision energy to determine the optimal mass spectrometry parameters for the characteristic peptides. A Scheduled MRM method was then established based on high-performance liquid chromatography-triple quadrupole mass spectrometry. Detection conditions are as follows:

[0030] Chromatographic conditions: The chromatographic column was an Xbridge Peptide BEH C18 (4.6 mm × 150 mm, 3.5 μm). Waters Corporation); Mobile phase A: 98% water - 2% acetonitrile - 0.1% formic acid; Mobile phase B: 98% acetonitrile - 2% water - 0.1% formic acid; Flow rate: 0.4 mL / min; Column temperature: 40 °C; Injection volume: 10 μL; Gradient elution program: 0.1–1 min, 3% B; 1–10 min, 3%–30% B; 10–13 min, 30%–55% B; 13–13.1 min, 55%–80% B; 13.1–16 min, 80% B; 16–16.1 min, 80%–3% B; 16.1–20 min, 3% B.

[0031] Mass spectrometry conditions: ESI+ positive ion mode; curtain gas 35 psi; nebulizer gas 65 psi; collision gas Medium; ionization voltage 4500 V; ion source temperature 500 °C; auxiliary gas 50 psi; positive ion scan Scheduled MRM mode, detection window 120 s, scan time 3 s.

[0032] Table 5. Information on Characteristic Peptides of Sesame Allergen Protein

[0033]

[0034]

[0035] This invention further relates to the use of nine characteristic peptides with processing stability in the detection of seven sesame allergenic proteins in processed foods. The nine characteristic peptides are as follows:

[0036] One Ses i 1 characteristic peptide: YLSQGR;

[0037] One Ses i 2 characteristic peptide: HCMQWMR;

[0038] One Ses i 3 characteristic peptide: GTISLVR;

[0039] One Ses i 4 characteristic peptide: QAGEAIK;

[0040] One Ses i 5 characteristic peptide: APHLQLQPR;

[0041] Ses i 6 has two characteristic peptides: GLIVMAR and ISGAQPSLR;

[0042] Ses i 7 has two characteristic peptides: VFSSTSR and NVIQPR.

[0043] The beneficial effects of this invention are:

[0044] (1) In this invention, 12 processed sesame products were used as samples, and 9 characteristic peptides of 7 sesame allergens were screened using ultra-high performance liquid chromatography-time-of-flight high-resolution mass spectrometry and ProteinPilot software. These characteristic peptides not only have good specificity, but also have processing stability.

[0045] (2) This invention optimizes the mass spectrometry parameters of characteristic peptides of crude sesame protein, improves their peak response intensity, and establishes a scheduled MRM method for nine characteristic peptides of sesame allergens. This method enables high-throughput and high-sensitivity detection of sesame allergens in processed foods, with accurate and stable results. Attached Figure Description

[0046] Figure 1 These are 12 sesame processed products prepared in a laboratory simulation.

[0047] Figure 2 This is a total ion current chromatogram of nine characteristic peptide segments of sesame allergen protein.

[0048] Figure 3 These are the detection results of 9 characteristic peptides in 12 sesame processed products. A: Detection results of characteristic peptides Ses i 1, Ses i 6, and Ses i 7; B: Detection results of characteristic peptides Ses i 2-Ses i 5.

[0049] Figures 4-6 This is a secondary mass spectrum of nine characteristic peptides from seven sesame allergen proteins.

[0050] Figure 7 This is a positive sample specificity verification result for 9 characteristic peptides of 7 sesame allergen proteins.

[0051] Figure 8 These are the stability results for nine characteristic peptides from seven sesame allergens. A: Baked at 120℃; B: Baked at 150℃; C: Baked at 180℃. Detailed Implementation

[0052] The present invention will be further illustrated by way of examples, but the present invention is not limited to the following examples.

[0053] Example 1:

[0054] (1) Laboratory simulation of sesame product preparation:

[0055] a. Stir-frying: Rinse black / white sesame seeds with water and drain. Stir-fry over medium-low heat for about 10 minutes, until the sesame seeds are plump. This will give you stir-fried and cooked white sesame seeds. Figure 1 A1) and black sesame ( Figure 1 (A2).

[0056] b. Grinding: After roasting, put the black / white sesame seeds into a grinder and grind them into a paste to obtain white sesame paste. Figure 1 B1) and black sesame paste ( Figure 1 (B2).

[0057] c. Baking: Add black / white sesame seeds to the low-gluten flour, press into cookie shapes, and bake in the oven (180℃, 20 minutes). You will get white sesame cookies. Figure 1 D1) and black sesame biscuits ( Figure 1 (D2).

[0058] d. Boiling: Grind roasted black / white sesame seeds and wrap them with glutinous rice dough to form glutinous rice balls. Boil in water over low heat until they float, about 5 minutes, to obtain boiled white sesame glutinous rice balls. Figure 1 F1) and black sesame glutinous rice balls ( Figure 1 (F2).

[0059] e. Steaming: Grind roasted black / white sesame seeds into powder, wrap them with all-purpose dough to form sesame buns, and steam for 15 minutes to obtain white sesame buns. Figure 1 E1) and black sesame buns ( Figure 1 (E2).

[0060] f. Deep-frying: Coat the glutinous rice dough with black / white sesame seeds to form sesame balls. Deep-fry at 150℃ over medium heat until golden brown, then fry at 180℃ for 1 minute to set, resulting in white sesame balls. Figure 1 C1) and black sesame balls ( Figure 1 (C2).

[0061] (2) Sample defatting: Take about 30g of sesame sample and place it in a mortar. Add liquid nitrogen to freeze it, and then grind it in a tissue grinder. Add hexane at a ratio of sesame:hexane = 1:5, stir at room temperature for 20 minutes, centrifuge (4000rpm, 10 minutes), discard the supernatant, and repeat the defatting process three times with the addition of hexane. After the last centrifugation, discard the supernatant, place the precipitate in a fume hood, and leave it overnight to allow the hexane to evaporate completely. Defatted sesame powder can be stored in a -80℃ refrigerator.

[0062] (3) Extraction of crude sesame protein: Weigh 0.5g of defatted sesame powder into a 15mL centrifuge tube, add 5mL of protein extraction buffer solution (50mM Tris-HCl + 7M urea + 2M thiourea + 4% CHAPS, pH 8.5), vortex for 1min, extract by shaking at room temperature for 20min, and then sonicate in a water bath for 20min. After protein extraction, centrifuge (12000rpm, 20min), filter the supernatant through a 0.22μm filter membrane, and use a Qubit filter. TM The kit was used to determine the protein concentration of the crude extract.

[0063] (4) Protein reduction alkylation: Based on the determined protein concentration, take approximately 200 μg of protein into a centrifuge tube and add an appropriate volume of mass spectrometry-graded water to a total volume of 50 μL. Add 10 μL of 120 mM DTT solution and incubate in a water bath (37 °C, 1 h). After cooling to room temperature, add 10 μL of 600 mM IAA solution and incubate in the dark for 15 min to complete the protein alkylation reaction.

[0064] (5) Trypsin digestion: Transfer the liquid in the centrifuge tube to a 5 kDa ultrafiltration membrane, centrifuge (12000 rpm, 20 min), add 100 μL of 50 mM ABC solution, centrifuge (12000 rpm, 15 min), repeat three times. Discard the liquid in the ultrafiltration tube, wash three times with mass spectrometer-grade water, and spin dry. Add 100 μL of 50 mM ABC solution, add 4 μL of 1 μg / μL trypsin at a 1:50 mass ratio, mix well, and incubate at 37℃ for 8 h. Remove the ultrafiltration tube, centrifuge (12000 rpm, 20 min), add 100 μL of 25 mM ABC solution, centrifuge (12000 rpm, 15 min), repeat three times to allow the enzyme-digested peptides on the ultrafiltration membrane to enter the filtrate. Remove the ultrafiltration membrane, place the ultrafiltration tube in a vacuum rotary evaporator for rotary drying, add 100-200 μL of mass spectrometer-grade water and continue rotary drying, repeat 3 times. Add 100 μL of mobile phase A (98% water - 2% acetonitrile - 0.1% formic acid), vortex to mix, centrifuge (10000 rpm, 10 min), and transfer 80 μL of liquid to a liquid chromatography vial. It can be stored in a refrigerator at 4°C for a short period of time.

[0065] (6) Ultra-high performance liquid chromatography-time-of-flight high-resolution mass spectrometry analysis: A liquid chromatography vial containing hydrolyzed peptides was placed in a sample tray. A mass spectrometry method was established for sample acquisition, and high-resolution analysis was performed. The determination conditions are as follows:

[0066] Chromatographic conditions: The chromatographic column was an Xbridge Peptide BEH C18 (4.6 mm × 150 mm, 3.5 μm). Waters Corporation); Mobile phase A: 98% water - 2% acetonitrile - 0.1% formic acid; Mobile phase B: 98% acetonitrile - 2% water - 0.1% formic acid; Flow rate: 0.25 mL / min; Column temperature: 40 °C; Injection volume: 5 μL; Gradient elution program: 0–0.5 min, 5%–8% B; 0.5–0.6 min, 8%–12% B; 0.6–25 min, 12%–30% B; 25–30 min, 30%–35% B; 30–30.5 min, 35%–80% B; 30.5–38 min, 80% B; 38–38.5 min, 80%–5% B; 38.5–50 min, 5% B.

[0067] Mass spectrometry conditions: Nanospray III ion source; ESI+ positive ion mode; spray voltage 2500V; nebulizer gas 6psi; curtain gas 30psi; temperature 150℃; information-dependent acquisition mode; TOF MS mode 350~1500m / z, 250ms; TOFMS / MS mode 100~1500m / z, 100ms, precursor ion charge range +2~+5; dynamic exclusion time 20s; cycle time 2s; Rolling CE enabled, runtime 50min.

[0068] (7) ProteinPilot software targeted identification: The data files obtained from high-resolution mass spectrometry were imported into ProteinPilot software. The amino acid sequence of sesame allergen protein was downloaded from the Uniprot database and used as the ProteinPilot database for identification of enzymatically digested peptides. The parameter settings were as follows: Sample type: identification; Cys alkylation: iodoacetamide; digestion: trypsin; search: fast ID; ID focus: biomodification; fixed modification: carbamoyl methylation; variable modification: methionine oxidation; peptide bias: ±0.05 Da ； MS / MS ion bias: ±0.03 Da ； Maximum allowable missed cleavage: 2. Select peptides with high mass spectrometry response intensity, 6-15 amino acids, mass-to-charge ratio <1250, confidence >95%, no missed cleavage sites, no non-enzymatic cleavage breaks, and no variable modifications as pre-selected characteristic peptides.

[0069] (8) Specificity verification of characteristic peptides: Open the Blast tool in the Uniprot database, input the pre-selected characteristic peptides for specificity analysis, and retain peptides whose species origin is only sesame. Protein extraction, trypsin digestion, and mass spectrometry analysis were performed on samples of barley, wheat, buckwheat, corn, rice, soybean, chickpea, peanut, pistachio, macadamia nut, cashew, pecan, almond, apricot kernel, pine nut, sunflower seed, walnut, hickory, cocoa bean, chestnut, Brazil nut, hazelnut, and different varieties of sesame to complete the specificity verification of characteristic peptides.

[0070] (9) Stability verification of characteristic peptides: Sesame seeds were baked at 120℃, 150℃ and 180℃ for 20 min, and then the stability of 9 characteristic peptides was verified by the Scheduled MRM method.

[0071] (10) Skyline software analysis: The amino acid sequence of sesame allergen protein was imported into Skyline software in FAST file format to establish a background library. The data obtained from high-resolution mass spectrometry was imported into Skyline as a library in WIF file format. The characteristic peptide sequence was entered on the left side of the Skyline page. The theoretical values ​​of enzyme digestion were compared with the established library, and the actual detected ion pairs were retained. The characteristic peptide ion pair information was exported.

[0072] (11) Establishment of Scheduled MRM Method: A Scheduled MRM method for mass spectrometry analysis was established based on characteristic peptide ion pair information, and sample acquisition methods were set for the determination of sesame allergen protein. The high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS) conditions are as follows:

[0073] Chromatographic conditions: The chromatographic column was an Xbridge Peptide BEH C18 (4.6 mm × 150 mm, 3.5 μm). Waters Corporation); Mobile phase A: 98% water - 2% acetonitrile - 0.1% formic acid; Mobile phase B: 98% acetonitrile - 2% water - 0.1% formic acid; Flow rate: 0.4 mL / min; Column temperature: 40 °C; Injection volume: 10 μL; Gradient elution program: 0.1–1 min, 3% B; 1–10 min, 3%–30% B; 10–13 min, 30%–55% B; 13–13.1 min, 55%–80% B; 13.1–16 min, 80% B; 16–16.1 min, 80%–3% B; 16.1–20 min, 3% B.

[0074] Mass spectrometry conditions: ESI+ positive ion mode; curtain gas 35 psi; nebulizer gas 65 psi; collision gas Medium; ionization voltage 4500 V; ion source temperature 500 °C; auxiliary gas 50 psi; positive ion scan Scheduled MRM mode, detection window 120 s, scan time 3 s.

[0075] (12) Mass spectrometry parameter optimization: The nine characteristic peptides were imported into the open-source software Skyline in the sky file format to obtain the theoretical DP and CE values ​​of these peptides. Based on the theoretical CE values, with a step size of 2 volts, 11 CE values ​​were set: theoretical CE, CE±10, CE±8, CE±6, CE±4, and CE±2. Following the CE value optimization, based on the theoretical DP values, with a step size of 10 volts, the optimization range was 20 to 130, and a total of 11 DP values ​​were set. The optimal DP and CE values ​​for each ion pair were determined according to the response intensity of the characteristic peptides.

[0076] The above description is merely one embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and improvements made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for detecting seven sesame allergen proteins in processed food, characterized in that, comprising the following steps: (1) sesame crude protein extraction: defatting the sesame sample, adding a protein extraction buffer solution, and extracting by oscillation combined with water bath ultrasonic assistance to obtain a sesame crude protein extract; (2) protein reduction and alkylation reaction: according to the determination of protein concentration, 200 μg of protein is taken in a centrifuge tube, an appropriate volume of mass spectrometry water is added to a total volume of 50 μL, 10 μL of 120 mM DTT solution is added, and water bath 37℃ for 1 h, after cooling to room temperature, 10 μL of 600 mM IAA solution is added to avoid light for 15 min, and the protein alkylation reaction is completed; (3) trypsin digestion: trypsin digestion is used to obtain hydrolyzed peptides; (4) analysis of hydrolyzed peptides based on ultra-high performance liquid chromatography-time-of-flight high resolution mass spectrometry: determine the chromatography and mass spectrometry conditions to analyze the hydrolyzed peptides; (5) characteristic peptide screening: the hydrolyzed peptides are identified by ProteinPilot software targeted analysis, and 9 characteristic peptides with processing stability in the seven sesame allergen proteins are determined by specific verification; (6) Scheduled MRM method establishment: mass spectrometry parameters are optimized for the ion pairs of the 9 sesame allergen characteristic peptides, and a Scheduled MRM method is established based on high performance liquid chromatography-triple quadrupole mass spectrometry to realize the detection of seven sesame allergen proteins in processed food; The 9 sesame allergen protein characteristic peptides are as follows: Ses i 1 characteristic peptide 1: YLSQGR; Ses i 2 characteristic peptide 1: HCMQWMR; Ses i 3 characteristic peptide 1: GTISLVR; Ses i 4 characteristic peptide 1: QAGEAIK; Ses i 5 characteristic peptide 1: APHLQLQPR; Ses i 6 characteristic peptide 2: GLIVMAR and ISGAQPSLR; Ses i 7 characteristic peptide 2: VFSSTSR and NVIQPR.

2. The method of claim 1, wherein the sesame sample in step (1) is a product prepared by one or a combination of processing processes such as tumbling, grinding, boiling, baking, frying and steaming, and the protein extraction buffer solution contains 7M urea, 2M thiourea and 4% CHAPS in 50mM Tris-HCl, pH 8.

5.

3. The method of claim 1, wherein step (4) analyzes the hydrolyzed peptides based on ultra-high performance liquid chromatography-time-of-flight high resolution mass spectrometry, and the determination conditions are as follows: ​ Chromatographic conditions: the chromatographic column was Xbridge Peptide BEH C18 4.6 mm x 150 mm, 3.5 μm, 300 Å, Waters Corporation; the mobile phase A was 98% water-2% acetonitrile-0.1% formic acid; the mobile phase B was 98% acetonitrile-2% water-0.1% formic acid; the flow rate was 0.25 mL / min; the column temperature was 40°C; the injection volume was 5 μL; the gradient elution program was as follows: 0-0.5 min, 5%-8% B; 0.5-0.6 min, 8%-12% B; 0.6-25 min, 12%-30% B; 25-30 min, 30%-35% B; 30-30.5 min, 35%-80% B; 30.5-38 min, 80% B; 38-38.5 min, 80%-5% B; 38.5-50 min, 5% B; Mass spectrometric conditions: Nanospray III ion source; ESI+ positive ion mode; spray voltage 2500 V; atomization gas 6 psi; curtain gas 30 psi; temperature 150°C; information-dependent acquisition mode; TOF MS mode 350-1500 m / z, 250 ms; TOF MS / MS mode 100-1500 m / z, 100 ms, parent ion charge range +2 to +5; dynamic exclusion time 20 s; cycle time 2 s; Rolling CE enabled, running time 50 min.

4. The method of claim 1, wherein the characteristic peptide segments are identified by hydrolysis of the peptide segments and targeted analysis by Protein Pilot software, and wherein the peptide segments with high response intensity, 6-15 amino acids, no missed cleavage site, and no non-enzymatic cleavage are selected as pre-selected characteristic peptide segments, and wherein the specificity of the peptide segments is analyzed by BLAST and verified by using sesame of different varieties and common food allergens as samples.

5. The method of claim 1, wherein the ion pairs, retention time, de-clustering voltage and collision energy of the characteristic peptide segments required for establishing the MRM method are as follows: 。 6. The method of claim 1, wherein step (6) is performed by high performance liquid chromatography-triple quadrupole mass spectrometry in a Scheduled MRM mode, and wherein the conditions are as follows: Chromatographic conditions: the chromatographic column was Xbridge Peptide BEH C18 4.6 mm x 150 mm, 3.5 μm, 300 Å, Waters Corporation; the mobile phase A was 98% water-2% acetonitrile-0.1% formic acid; the mobile phase B was 98% acetonitrile-2% water-0.1% formic acid; the flow rate was 0.4 mL / min; the column temperature was 40°C; the injection volume was 10 μL; the gradient elution program was as follows: 0.1-1 min, 3% B; 1-10 min, 3%-30% B; 10-13 min, 30%-55% B, 13-13.1 min, 55%-80% B, 13.1-16 min, 80% B, 16-16.1 min, 80%-3%, 16.1-20 min, 3% B; Mass spectrometry conditions: ESI+ positive ion mode; gas curtain gas 35 psi; atomizing gas 65 psi; collision gas Medium; ionization voltage 4500 V; ion source temperature 500℃; auxiliary gas 50 psi; positive ion scan Scheduled MRM mode, detection window 120 s, scan time 3 s.

7. Use of nine characteristic peptide segments with process stability for the detection of seven sesame allergen proteins in processed food, characterized by The 9 characteristic peptide segments are as follows: 1 Ses i 1 characteristic peptide segment: YLSQGR; 1 Ses i 2 characteristic peptide segment: HCMQWMR; 1 Ses i 3 characteristic peptide segment: GTISLVR; 1 Ses i 4 characteristic peptide segment: QAGEAIK; 1 Ses i 5 characteristic peptide segment: APHLQLQPR; 2 Ses i 6 characteristic peptide segments: GLIVMAR and ISGAQPSLR; 2 Ses i 7 characteristic peptide segments: VFSSTSR and NVIQPR; The 9 characteristic peptide segments are determined by screening according to any one of claims 1-6.

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