CN112662726A - Identification and detection method of allergen protein T cell epitope polypeptide - Google Patents
Identification and detection method of allergen protein T cell epitope polypeptide Download PDFInfo
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Abstract
The invention discloses an identification and detection method of allergen protein T cell epitope polypeptide, belonging to the technical field of identification and evaluation of allergen protein T cell epitope polypeptide. The invention utilizes liquid chromatography tandem mass spectrometry to identify and detect the polypeptide generated by degrading the allergen protein by the dendritic cell, and evaluates the T cell epitope polypeptide by constructing a T lymphocyte model. The method can comprehensively reflect degradation polypeptide products of the allergen protein in the dendritic cells and the relation between the polypeptide and the epitope, can be used for evaluating the allergenicity change of the allergen protein in the sensitization stage, has high sensitivity, high accuracy and high precision, and is suitable for the identification of all types of allergen protein and epitope polypeptide degraded by the dendritic cells.
Description
Technical Field
The invention belongs to the technical field of identification and evaluation of allergen protein T cell epitope polypeptides, and particularly relates to an identification and detection method of allergen protein T cell epitope polypeptides.
Background
At present, food allergy has become a food safety problem of great concern at home and abroad. Food allergy is an immunoglobulin e (ige) -mediated type I (immediate) hypersensitivity reaction. In the sensitization stage, antigen enters the body for the first time, is taken up by antigen presenting cells and then presents to helper T lymphocytes (Th cells), so that the Th cells activate initial Th0 cells to differentiate to Th2 cells, and cytokines secreted by Th2 cells, such as interleukin-4, interleukin-13 and the like, can induce B lymphocytes to activate and secrete antigen-specific IgE antibodies. Specific IgE antibodies bind to the high affinity receptor fceri on the surface of effector cells (e.g., mast cells, basophils, etc.). In the effect stage, the antigen enters the body again and is combined with IgE on the surface of an effector cell, so that the degranulation phenomenon is promoted, and histamine, tryptase and other media are released to enable the body to generate anaphylactic reaction.
Dendritic Cells (DCs) are the most important antigen presenting cells, and immature DCs have very active non-specific phagocytic function. Antigens are phagocytosed by DCs and degraded by endoplasmic reticulum enzyme systems into peptide fragments, which are presented to the cell surface, binding to specific receptors on the surface of T cells, stimulating initial Th 0-type cell polarization. Therefore, the detection and identification of degradation products of allergen proteins in dendritic cells is of great importance for the assessment of food allergy, especially in the sensitization phase. At present, few documents report how to detect polypeptides produced by dendritic cell degradation. With the development of the biological mass spectrometry technology, the method can accurately detect trace substances, and can avoid the problems of false positive, matrix interference and the like faced by common detection methods such as a Polymerase Chain Reaction (PCR) method, an enzyme-linked immunosorbent assay (ELISA) method and the like. The liquid chromatography tandem mass spectrometry is used for separating various components in a sample by liquid phase and analyzing peptide fragments degraded by the allergen by using an ionization technology.
Epitopes are also called antigenic determinants and are divided into B-cell epitopes and T-cell epitopes. T cell epitopes are linear peptide fragments presented by the antigen to the T cell surface antigen receptor by the histocompatibility complex (MHC) molecules after the antigen has been treated by the antigen presenting cell, and play an important role in the initiation and regulation of specific immune responses. Therefore, the method for identifying whether the polypeptide generated by degrading the antigen protein by the dendritic cells is the T cell epitope or not has important significance for the food allergen allergenicity evaluation method. T cell epitope screening methods mainly include direct methods and indirect methods. The direct method elutes the polypeptide combined with MHC molecules through acid treatment, and mass spectrometry is carried out after purification; the indirect method is to synthesize a peptide library overlapping with a defined antigen, and a polypeptide capable of producing an effect of activating proliferation by lymphocytes of an animal or human immunized with the protein is defined as a T cell epitope. At present, the identification of allergens, especially T cell epitopes of small molecule proteins, mainly adopts an indirect method.
The main methods for detecting and identifying the polypeptide generated by the degradation of dendritic cells in the prior art are as follows: extracting endoplasmic reticulum enzyme system from dendritic cell or JAWSII cell line, co-culturing with allergen protein in vitro, and detecting degraded polypeptide product by mass spectrum. In the prior art, microsomes separated from cells and allergen proteins are co-cultured, and polypeptide products degraded by the allergen proteins under the action of the microsomes are detected. The main defects are that the activity of an endoplasmic reticulum enzyme system in microsomes extracted from cells is different from the activity of the endoplasmic reticulum enzyme system in the cells, so that the dendritic cells cannot be reduced to phagocytose the cells really, and the endoplasmic reticulum enzyme system degrades allergen proteins. In addition, the prior art does not validate the degraded polypeptides, only by database matching.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for identifying and detecting an allergen protein T cell epitope polypeptide. The method adopts a liquid phase tandem mass spectrometry method to detect the polypeptide of the allergen protein degraded by the dendritic cells and verify the antigen epitope polypeptide. The method can comprehensively reflect degradation polypeptide products of the allergen protein in dendritic cells and the relation between the polypeptide and the epitope, and can be used for evaluating the sensitization change of the allergen protein in a sensitization stage.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for identifying and detecting allergen protein T cell epitope polypeptide comprises the following steps:
(1) polypeptide for detecting degradation of dendritic cells by constructing liquid chromatography-tandem mass spectrometry method
Carrying out ultrafiltration tube centrifugation on the supernatant obtained after the co-culture of the allergen protein and the dendritic cells, and carrying out UHPLC-MS/MS detection after desalting treatment on the collected filtrate; searching an allergen Protein amino acid sequence in an NCBI Protein library to establish a database, searching and comparing data detected by UHPLC-MS/MS in the established database by adopting Protein Pilot software to obtain a polypeptide sequence of dendritic cells after degrading the allergen Protein; synthesizing the degraded polypeptide for T cell epitope identification;
(2) constructing T lymphocyte model aiming at allergen protein, and identifying epitope polypeptide
Adding the polypeptides synthesized in the step (1) into T lymphocytes aiming at allergen proteins respectively, detecting the proliferation condition of the T lymphocytes, and considering the polypeptides capable of causing the T lymphocytes to proliferate as T cell epitope polypeptides, otherwise, the polypeptides are common peptide fragments;
(3) construction of Q-trap method for detecting the T cell epitope polypeptide
Synthesizing the T cell epitope polypeptide identified in the step (2) as a standard product, and constructing a Q-trap method for detection.
On the basis of the above scheme, in the step (1), the allergen protein and the dendritic cells are cultured together as follows: dendritic cells were adjusted to a density of 2X 106suspension/mL, adding allergen protein to dendritic cell suspension at 10. mu.g/mL in 5% CO2Cultivation in incubator 3After 0min, the reaction was stopped by adding pre-cooled PBS.
On the basis of the scheme, in the UHPLC-MS/MS detection in the step (1), the polypeptides degraded by dendritic cells are detected by using a UPLC-Q-TOF data-dependent acquisition analysis mode (DDA) and a data-independent acquisition analysis mode (DIA).
On the basis of the scheme, the parameters for performing library searching comparison by adopting Protein Pilot software in the step (1) are set as follows: digestion mode: none; peptide fragment selection conditions were: the confidence coefficient is more than 90 percent; the FDR value is less than or equal to 1 percent; ③ it is not modified.
On the basis of the scheme, the dendritic cells are any one of immortalized cell lines and primary cells;
preferably, the immortalized cell line is a mouse myeloid dendritic cell line (DC 2.4); the primary cell is any one of spleen or peripheral blood derived dendritic cells, other strains of mouse (C57 and the like) derived dendritic cells and human peripheral blood derived dendritic cells.
On the basis of the scheme, the T lymphocyte is any one of an immortalized cell line and a primary cell; the immortalized cell line is an antigen-specific T lymphocyte cell line; the primary cell is any one of mouse spleen T lymphocyte, mesenteric lymph node, Peyer's patch T lymphocyte and the like and human source peripheral blood mononuclear cell.
On the basis of the scheme, the allergen protein is any one of tropomyosin, parvalbumin, troponin and ovalbumin.
On the basis of the scheme, the Q-trap detection method in the step (3) comprises the following steps:
predicting MRM transmission ion pairs of T cell epitope polypeptides through Skyline, and optimizing collision energy and clustering voltage of parent ions; precursor ion charge number selection 2 or 3; ion number charge number selection 1, 2; the fragmentation type of the peptide fragment ions is b and y; the mass-to-charge ratio range of the daughter ions is 100-1250, and the ion pairs of the T cell epitope polypeptides are screened by searching and comparing results through contrast Protein Pilot;
liquid chromatographyConditions are as follows: a chromatographic column: the advanced bio Peptide Map column (150mm 2.1mm,
2.7 μm), column temperature: 40 ℃; sample introduction amount: 20 mu L of the solution; mobile phase A: 0.1% formic acid-water, mobile phase B: 0.1% formic acid-acetonitrile, flow rate: 0.35mL/min, gradient elution procedure as follows:
| time (min) | Mobile phase A | |
| 0 | 95 | 5 |
| 0.5 | 65 | 35 |
| 2 | 50 | 50 |
| 6 | 95 | 5 |
| 8 | 95 | 5 |
The mass spectrum conditions are as follows: electrospray positive ion (ESI +) mode; spraying voltage: 5500V; setting ion source parameters: atomizing gas pressure: 60psi, auxiliary heating gas pressure: 50psi, air curtain pressure: 35psi, ion source temperature: 575 ℃.
The invention has the following beneficial effects:
1. the method directly uses the allergen protein and the dendritic cells for co-culture, so that the scenes of reduction, phagocytosis and degradation are relatively real; on the basis, a liquid-phase tandem mass spectrometry method is established, and the polypeptide generated by degrading the allergen protein by the dendritic cells is detected through a data-dependent acquisition analysis mode (DDA) and a data-independent acquisition analysis mode (DIA), and the antigen epitope polypeptide is identified and verified. The method can comprehensively react degradation products of the allergen protein in dendritic cells through proteomic non-targeted analysis, verifies the relation between degradation polypeptides and antigen epitopes through cytology experiments, and can be used for evaluating the sensitization change of the allergen protein in a sensitization stage.
2. The method has the advantages of high sensitivity, high accuracy and high precision, and is suitable for identifying all types of allergen proteins and epitope polypeptides degraded by dendritic cells.
3. In one embodiment, the present invention provides a method for identifying a substance comprising polypeptide 1(VQESLLKANIQLVEK) in a sample by synthesizing the substance as a label. The method is validated against a target polypeptide and is suitable for identifying polypeptides having a defined amino acid sequence. The peptide fragment is verified by a subsequent T cell proliferation experiment and is a T cell epitope polypeptide of the allergen protein.
4. The invention adopts the polypeptide 2(EERLNTATTKLAEAS), the polypeptide 3(NDLDQVQESL) and the polypeptide 4(LLEEDLERS) as qualitative peptide fragments to identify whether a sample contains the polypeptide substance. The method is validated against a target polypeptide and is suitable for identifying polypeptides having a defined amino acid sequence.
Drawings
FIG. 1 is a flow assay of dendritic cells;
FIG. 2 is a mass spectrometric analysis of degradation products-DDA of dendritic cells co-cultured with allergen proteins for 30 min;
FIG. 3 is mass spectrometry analysis of degradation products-DIA of dendritic cells incubated with allergen proteins for 30 min;
FIG. 4 shows the detection pattern of polypeptide 1(VQESLLKANIQLVEK) -DIA detection in a sample;
FIG. 5 shows the detection pattern of polypeptide 2(EERLNTATTKLAEAS) -DIA detection in a sample;
FIG. 6 shows the detection pattern of polypeptide 3(NDLDQVQESL) -DIA detection in a sample;
FIG. 7 shows the detection pattern of polypeptide 4(LLEEDLERS) -DIA detection in a sample;
FIG. 8 is a CSFE-tagged synthetic polypeptide stimulating mouse spleen T lymphocytes;
FIG. 9 is a polypeptide standard-polypeptide 1(VQESLLKANIQLVEK) Q-trap detection map;
FIG. 10 is a diagram of the Q-trap detection of polypeptide 1(VQESLLKANIQLVEK) in a sample.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Taking a mouse as an example, the invention detects the polypeptide generated by degrading the allergen protein by dendritic cells by a liquid chromatography tandem mass spectrometry method, analyzes the relationship between the polypeptide generated by degrading and antigen epitope by utilizing spleen T lymphocytes of the mouse immunized by the allergen protein, evaluates the relationship between the degradation product and the antigen epitope of the allergen protein after the allergen protein is phagocytized by the dendritic cells and an endoplasmic reticulum enzyme system, and indirectly characterizes the allergenicity change of the allergen protein at the sensitization stage, and mainly comprises the following steps:
(1) extracting and culturing the dendritic cells of the mouse medullary system;
(2) co-culturing the allergen protein and dendritic cells, and evaluating the degradation condition of the allergen protein in the dendritic cells;
(3) constructing a liquid chromatography tandem mass spectrometry method to detect the polypeptide generated by degradation;
(4) constructing a T lymphocyte model of an allergen protein immune mouse, and identifying epitope polypeptide;
(5) constructing a method for detecting a target polypeptide by using Q-trap.
Example 1
(1) Extraction and culture of mouse medullary dendritic cells
BALB/C female mice of 6 weeks of age were selected, anesthetized, decapped, sacrificed, soaked in 75% ethanol for 3-5 minutes, and hind-limb femurs and tibiae were aseptically separated, placed in petri dishes, and washed 2 times with pre-cooled PBS. The pre-cooled RPMI-1640 medium was aspirated by a 1mL disposable syringe, and the two ends of the bone were pierced into the bone marrow cavity and washed repeatedly until the bone marrow cavity became white. The washed bone marrow was aspirated and filtered through a 70 μm cell sieve. The bone marrow cell suspension was collected into a 15mL centrifuge tube and centrifuged (600g, 10 min). Centrifuging, removing the supernatant, adding 5mL of erythrocyte lysate, blowing, uniformly mixing, standing at room temperature for 3min, centrifuging, and removing the supernatant. Washed 2 times with pre-cooled RPMI-1640 medium. Cell concentrations were adjusted to 2X 10 with RPMI-1640 complete medium (10% FBS + 1% double anti/anti mycoplasma, rm GM-CSF: 20ng/mL, rm IL-4: 10ng/mL)6Perml, seeded in 6-well plates, 4mL per well. Cells were exposed to 5% CO2After 48h incubation in the incubator, the entire medium was aspirated and the same volume of complete medium was added. And (5) changing the liquid at the 5 th and 6 th semi-quantitative times, culturing to the 7 th, and slightly blowing and collecting all the suspension cells. The cells were washed 2 times with PBS and adjusted to a cell concentration of 2X 106and/mL, adding FITC-cd11c flow dye, reacting for 30min at 4 ℃ in a dark place, washing twice with PBS, removing supernatant, resuspending, and performing flow detection. The detection result is shown in fig. 1, the purity of the dendritic cells is 88.43%, the requirement of the experiment is met, and the next experiment can be carried out.
(2) Co-culture of allergen protein-tropomyosin with dendritic cells
The dendritic cell density was adjusted to 2X 106Perml/mL in 24-well plates, 10. mu.g allergen protein (tropomyosin) per mL of cells, and placing the 24-well plates in 5% CO2Culturing in incubator for 30min, adding 200 μ L of precooledPBS terminates dendritic cell phagocytic proteins. The supernatant was collected by centrifugation into an EP tube, which was quickly placed in a liquid nitrogen tank, and the sample was then transferred to a-80 ℃ freezer for long-term storage.
(3) Method for constructing liquid chromatogram tandem mass spectrum detection
The supernatant of the dendritic cell-degraded allergen protein was centrifuged by using a 3kDa ultrafiltration tube (10000rpm, 20min), the filtrate was collected in a new EP tube, and the sample was desalted by using a ZipTip C18 column: the micro-column was washed 5 times with acetonitrile, and the micro-column was activated with 50% acetonitrile-water and equilibrated with 0.1% formic acid. Sucking the filtrate, and repeatedly sucking and beating for 20-30 times. The column was washed with 0.1% formic acid 3 times for desalting treatment, followed by gradient elution with 20. mu.L of 0.1% formic acid in 20% acetonitrile-water, 20. mu.L of 0.1% formic acid in 50% acetonitrile-water, and 20. mu.L of 0.1% formic acid in 70% acetonitrile-water, respectively, and the eluates were collected in a liquid phase vial. Then 100. mu.L of 0.1% formic acid was added to the liquid phase vial for UHPLC-MS/MS detection. The results are shown in FIG. 2, in which allergen protein and dendritic cell are co-cultured for 30min, and the distinct chromatographic peaks at 24.69,28.24,31.50,36.28,48.45min are detected. FIG. 3 is mass spectrometry analysis of degradation products-DIA of dendritic cells incubated with allergen proteins for 30 min.
Wherein, UHPLC-MS/MS adopts two scanning modes of UPLC-Q-TOF data dependent acquisition analysis (DDA) and data independent acquisition analysis (DIA).
The data is dependent on the acquisition analysis mode, and the conditions are set as follows:
liquid chromatography conditions: the chromatographic column was an agilent advanced bio Peptide Map column (150mm x 2.1mm,
2.7 μm); column temperature: at 40 ℃. Mobile phase A: 0.1% formic acid-acetonitrile, mobile phase B: 0.1% formic acid-water; flow rate: 0.25 mL/min; sample introduction amount: 30 mu L of the solution; sample introduction time: 46 min; gradient of mobile phase: 0-2min (95% B), 2-17min (95% -80% B), 27-37min (85% -65% B)，37-39min(65％-20％B)，39-42min(20％-95％B)，42-46min(95％B)；
Mass spectrum conditions: electrospray positive ion (ESI +) mode; the spraying voltage is 5500V; mass TOF mass to charge ratio (m/z) scan range: 350-1500 Da; cumulative time: 0.2 s; IDA trigger condition: mass to charge ratio (m/z) trigger range: 350-1200Da, charge number: 2-4, intensity threshold: 150cps, quality accuracy threshold: 50mDa, again triggering the acquisition interval: 4 s; fragment ion acquisition mass to charge ratio (m/z) scan range: 100-1500 Da; cumulative time: 0.05 s; the strongest ion number monitored in each acquisition cycle: 20; selecting a high sensitivity mode; setting ion source parameters: atomizing gas GS 1: 60psi, supplemental heater GS 2: 50psi, gas curtain pressure 35psi, ion source temperature: 525 ℃, declustering voltage: 100V, ion scan collision energy: dynamically varying with the mass-to-charge ratio (m/z).
Searching allergen Protein amino acid sequences in an NCBI Protein library to establish a database (the south America white shrimp (Litopenaeus vannamei) is downloaded in 1 month 2020 and totally contains 632350 Protein sequences), and performing library searching comparison by adopting Protein Pilot software to search parameters: digestion mode: none, peptide fragment selection conditions were: (1) the confidence coefficient is more than 90 percent; (2) the FDR value is less than or equal to 1 percent; (3) it was not modified.
The polypeptide information of allergen protein degradation in dendritic cells is shown in table 1:
TABLE 1
The peptide fragments in table 1 were chemically synthesized (purity higher than 95%) for epitope identification.
The data is independent of the acquisition and analysis mode, and the conditions are set as follows:
selecting the most abundant (highest corresponding intensity) data in DDA analysis in a sample by using PeakView software, then opening a total ion flow graph, double-clicking to display fragment ion images, selecting an m/z 300-1250 range, opening a dialog box of mass-to-charge ratio and response intensity, exporting the data of the m/z 300-1250 range, and importing the data into a SWATH variable window calculator. The setup of the SWATH acquisition is as follows: and (4) target window: 60, mass to charge ratio range: 300-1250; window overlap width: 1 Da; minimum window width: 3 Da; collision energy expansion: 15V.
And (3) predicting MRM transmission ion pairs of the polypeptide through Skyline, and optimizing the collision energy and the clustering voltage of parent ions. Precursor ion charge number selection 2 or 3; ion number charge number selection 1, 2; the fragmentation type of the peptide fragment ions is b and y; the mass-to-charge ratio of the daughter ions is 100-1250, and the ion pairs of the polypeptide are screened by contrasting the Protein Pilot identification result. Wherein:
polypeptide 1(VQESLLKANIQLVEK), the parent ion of which is: 571.336605, the product ions are: 742.937773(3y13), 678.416477(3y12), 521.816398(3y9), 798.47198(3b 7);
polypeptide 2(EERLNTATTKLAEAS), the parent ion of which is: 409.2152, the product ions are: 415.1936(b3),528.2776(b4),642.3206(b5),458.2302(b8),508.7540(b9),618.3457(y6),490.2508(y5),496.7666(y10),410.7242(y 8);
polypeptide 3(NDLDQVQESL) whose parent ion 1 is: 580.7751, the product ions are: 586.2467(b5), 685.3151(b6), 813.3737(b7), 931.4731(y8), 818.3890(y7)703.3621(y 6); the parent ion 2 is: 387.5192, the product ions are: 458.1882(b4), 586.2467(b5), 685.3151(b6), 407.1905(b7)471.7118(b8), 515.2278(b9), 703.3621(y6), 575.3035(y5), 476.2351(y4), 523.7537(y9), 466.2402(y8), 409.6982(y 7);
polypeptide 4(LLEEDLERS) having a parent ion 1 of 552.2826 and daughter ions of: 600.2875(b5), 713.3716(b6), 842.4142(b7), 877.3898(y7), 748.3472(y6)619.3046(y 5); the parent ion 2 is: 368.5241, the product ions are: 485.2606(b4), 600.2875(b5), 713.3716(b6), 421.7107(b7), 499.7613(b8), 619.3046(y5), 504.2776(y4), 391.1985(y3)495.7406(y8), 439.1985(y7), 374.6772(y 6).
The DIA detection results are shown in fig. 4-7, where different bars in fig. 4-7 indicate different daughter ion intensities. Wherein FIG. 4, FIG. 5, FIG. 6 and FIG. 7 show the detection of polypeptide 1, polypeptide 2, polypeptide 3 and polypeptide 4, respectively, in a sample.
(4) And (3) constructing a T lymphocyte model of an allergen protein immune mouse, and identifying the epitope polypeptide.
An allergic mouse model is constructed by immunizing a BALB/C female mouse with allergen protein.
The method comprises the following specific steps:
BALB/C female mice of 6 weeks old, SPF grade, after adaptive feeding for 7 days, allergen protein injection was performed.
And (3) sensitization stage: intraperitoneal injection is carried out on 1d, 7d, 13d, 19d, 25d and 31d (50 mu g of allergen protein and aluminum hydroxide adjuvant, 1:3, v/v);
and (3) an excitation stage: intraperitoneal injection (100 μ g allergen protein) was performed at 34d, 37d, 40d, and 43d, respectively;
a challenge stage: intraperitoneal injection (1mg allergen protein) is carried out at 50d, cervical dislocation is carried out at 51d after anesthesia, spleen is taken out under sterile environment, a small amount of RPMI-1640 is added for grinding, a 70 mu m cell sieve is used, centrifugation (600g, 10min) is carried out, supernatant is discarded, 5mL of erythrocyte lysate is added, the mixture is blown to be uniform and kept stand for 5min at room temperature, and cells are washed twice by RPMI-1640 (containing 10% FBS). The cells were transferred to a 15mL centrifuge tube, washed twice with PBS and resuspended (5-10X 10)6/mL), 5. mu.M CFSE per mL of cells was incubated for 15min at room temperature in the dark with shaking at least once in between. Staining was stopped by incubation on ice for 5min with 5 volumes of RPMI-1640 (10% FBS). Cell washing with RPMI-1640 complete medium three times, resuspension and adjustment of cell concentration to 2X 106Perml, inoculated into 24-well plates, 200. mu.g of the synthesized polypeptide was added to each well, and the 24-well plates were placed in 5% CO2And (5) culturing in an incubator for 3 d. The cells were collected, washed twice with PBS and resuspended, and examined by flow cytometry, as shown in figure 8.
The result shows that the polypeptide VQESLLKANIQLVEK has obvious proliferation effect on T cells, and the proliferation rate is 26.2%, wherein NC is negative control, and ConA is positive control.
(5) Method for constructing Q-trap to detect target polypeptide
T cell epitope polypeptide (VQESLLKANIQLVEK) was selected for Q-trap detection by the above procedure. T-cell epitope polypeptides (VQESLLKANIQLVEK) were synthesized (greater than 98% pure) for detection of polypeptides in samples (cell supernatant sample-extracellular polypeptides and cell lysate sample-intracellular polypeptides).
And (3) predicting MRM transmission ion pairs of the polypeptide through Skyline, and optimizing the collision energy and the clustering voltage of parent ions. Precursor ion charge number selection 2 or 3; ion number charge number selection 1, 2; the fragmentation type of the peptide fragment ions is b and y; the mass-to-charge ratio of the daughter ions is 100-1250, and the ion pairs of the polypeptide are screened by contrasting the Protein Pilot identification result.
Wherein, the T cell epitope polypeptide (VQESLLKANIQLVEK) has parent ions of: 571.336605, the product ions are: 742.937773(3y13), 678.416477(3y12), 521.816398(3y9), 798.47198(3b 7).
Liquid chromatography conditions: a chromatographic column: the advanced bio Peptide Map column (150mm 2.1mm,
2.7 μm), column temperature: 40 ℃; sample introduction amount: 20 μ L. Mobile phase A: 0.1% formic acid-water, mobile phase B: 0.1% formic acid-acetonitrile, flow rate: 0.35mL/min, gradient elution procedure as follows: 0 min: mobile phase a, 95%, mobile phase B5%; 0.5 min: mobile phase a, 65%, mobile phase B35%; 2 min: mobile phase a, 50%, mobile phase B50%; 6 min: mobile phase a, 95%, mobile phase B5%; 8 min: mobile phase A, 95%, mobile phase B5%
The mass spectrum conditions are as follows: electrospray positive ion (ESI +) mode; spraying voltage: 5500V. Setting ion source parameters: atomizing gas pressure: 60psi, auxiliary heating gas pressure: 50psi, air curtain pressure: 35psi, ion source temperature: 575 ℃.
The detection results are shown in fig. 9 and 10. Wherein FIG. 9 shows a chromatogram of a polypeptide standard at a concentration of 2. mu.g/mL; FIG. 10 shows a chromatogram of polypeptides in a sample, with allergen proteins co-cultured with dendritic cells for 30 min. The content of the target polypeptide in the sample can be detected by the method.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method for identifying and detecting allergen protein T cell epitope polypeptide is characterized by comprising the following steps:
(1) polypeptide for detecting degradation of dendritic cells by constructing liquid chromatography-tandem mass spectrometry method
Carrying out ultrafiltration tube centrifugation on the supernatant obtained after the co-culture of the allergen protein and the dendritic cells, and carrying out UHPLC-MS/MS detection after desalting treatment on the collected filtrate; searching an allergen Protein amino acid sequence in an NCBI Protein library to establish a database, searching and comparing data detected by UHPLC-MS/MS in the established database by adopting Protein Pilot software to obtain a polypeptide sequence of the dendritic cells degrading the allergen Protein; synthesizing the degraded polypeptide for T cell epitope identification;
(2) constructing T lymphocyte model aiming at allergen protein, and identifying epitope polypeptide
Adding the polypeptides synthesized in the step (1) into T lymphocytes aiming at allergen proteins respectively, detecting the proliferation condition of the T lymphocytes, and considering the polypeptides capable of causing the T lymphocytes to proliferate as T cell epitope polypeptides, otherwise, the polypeptides are common peptide fragments;
(3) construction of Q-trap method for detecting the T cell epitope polypeptide
Synthesizing the T cell epitope polypeptide identified in the step (2) as a standard product, and constructing a Q-trap method for detection.
2. The method for identifying and detecting an allergen protein T-cell epitope polypeptide according to claim 1, wherein in step (1), the allergen protein is conjugated to a dendritic cellThe cell co-culture comprises the following steps: dendritic cells were adjusted to a density of 2X 106suspension/mL, adding allergen protein to dendritic cell suspension at 10. mu.g/mL in 5% CO2After incubation in an incubator for 30min, precooled PBS was added to stop the reaction.
3. The method for identifying and detecting an allergen protein T-cell epitope polypeptide according to claim 1, wherein in the UHPLC-MS/MS detection in step (1), a UPLC-Q-TOF data-dependent acquisition analysis mode and a data-independent acquisition analysis mode are used for detecting a polypeptide degraded by dendritic cells.
4. The method for identifying and detecting an allergen Protein T cell epitope polypeptide according to claim 1, wherein the parameters for performing library searching alignment in step (1) using Protein Pilot software are set as follows: digestion mode: none; peptide fragment selection conditions were: the confidence coefficient is more than 90 percent; the FDR value is less than or equal to 1 percent; ③ it is not modified.
5. The method for identifying and detecting an allergen protein T cell epitope polypeptide according to claim 1, wherein said dendritic cell is any one of an immortalized cell line and a primary cell.
6. The method for identifying and detecting an allergen protein T cell epitope polypeptide of claim 5, wherein said immortalized cell line is a mouse myeloid dendritic cell line; the primary cell is any one of spleen or peripheral blood derived dendritic cells, other strains of mouse derived dendritic cells and human peripheral blood derived dendritic cells.
7. The method for identifying and detecting an allergen protein T cell epitope polypeptide according to claim 1, wherein said T lymphocyte is any one of an immortalized cell line and a primary cell.
8. The method for identifying and detecting an allergen protein T cell epitope polypeptide of claim 7, wherein said immortalized cell line is an antigen-specific T lymphocyte cell line; the primary cell is any one of mouse spleen T lymphocyte, mesenteric lymph node and Peyer's lymph node T lymphocyte and human source peripheral blood mononuclear cell.
9. The method for identifying and detecting an allergen protein T-cell epitope polypeptide according to any one of claims 1 to 8, wherein the allergen protein is any one of allergenic proteins such as tropomyosin, parvalbumin, troponin, and ovalbumin.
10. The method for identifying and detecting an allergen protein T-cell epitope polypeptide according to claim 9, wherein the Q-trap detection method in step (3) is:
predicting MRM transmission ion pairs of T cell epitope polypeptides through Skyline, and optimizing collision energy and clustering voltage of parent ions; precursor ion charge number selection 2 or 3; ion number charge number selection 1, 2; the fragmentation type of the peptide fragment ions is b and y; the mass-to-charge ratio range of the daughter ions is 100-1250, and the ion pairs of the T cell epitope polypeptides are screened by searching and comparing results through contrast Protein Pilot;
liquid chromatography conditions: a chromatographic column: the advanced bio Peptide Map column (150mm 2.1mm,
2.7 μm), column temperature: 40 ℃; sample introduction amount: 20 mu L of the solution; mobile phase A: 0.1% formic acid-water, mobile phase B: 0.1% formic acid-acetonitrile, flow rate: 0.35mL/min, gradient elution procedure as follows:
The mass spectrum conditions are as follows: electrospray positive ion (ESI +) mode; spraying voltage: 5500V; setting ion source parameters: atomizing gas pressure: 60psi, auxiliary heating gas pressure: 50psi, air curtain pressure: 35psi, ion source temperature: 575 ℃.
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