CN116804047A - Method for detecting S protein content in novel coronavirus vaccine - Google Patents
Method for detecting S protein content in novel coronavirus vaccine Download PDFInfo
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- CN116804047A CN116804047A CN202210265988.9A CN202210265988A CN116804047A CN 116804047 A CN116804047 A CN 116804047A CN 202210265988 A CN202210265988 A CN 202210265988A CN 116804047 A CN116804047 A CN 116804047A
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Abstract
The present disclosure relates to methods for detecting the content of S protein in novel coronavirus vaccines. The present disclosure provides a polypeptide for quantitatively detecting a novel coronavirus S protein, the polypeptide comprising an amino acid sequence as shown in VGGNYNYLYR (SEQ ID NO: 1) or VGGNYYNYR (SEQ ID NO: 2). The disclosure also provides kits comprising the polypeptides, and methods for quantitatively detecting novel coronavirus S proteins.
Description
Technical Field
The present disclosure relates to the field of detection technology of novel coronavirus (SARS-CoV-2) inactivated vaccines. In particular to a characteristic polypeptide for detecting the S protein content of SARS-CoV-2 inactivated vaccine, a detection kit and a detection method thereof.
Background
The novel coronavirus pneumonia (COVID-19) is a disease with severe acute respiratory syndrome coronavirus (SARS-CoV-2) as a pathogen, and can cause acute respiratory infectious diseases of human bodies. In order to cope with spread of covd-19, vaccines are an effective way, which is a focus of global drug development.
Inactivated vaccines are the most effective vaccine development pathway against infectious diseases. Currently, the SARS-CoV-2 virus gradually evolves from its prototype strain into a number of major variants, including Alpha, beta, gamma, delta, omicron, etc. New generation variant vaccines are being developed and produced against the prevalence of these variants, as well as bivalent or multivalent vaccines against the prototype and variant strains. The method has very important significance for vaccine evaluation in terms of the content of effective antigens (immunogens) of different strains in the inactivated vaccine, such as the content detection of S protein and N protein in SARS-CoV-2.
The antigen content of a vaccine is usually quantified using an enzyme-linked immunosorbent assay (ELISA), which is one of the key evaluation indicators of the vaccine. However, since the virus epitopes of the prototype strain and the variant strain are not different, the prototype strain and the variant strain cannot be accurately quantified by an ELISA antigen detection system when evaluating the antigen content of bivalent or multivalent vaccine comprising the prototype strain and the variant strain. Thus, there is a need to establish a method that enables accurate quantification of different antigens in a bivalent or multivalent vaccine or intermediate, respectively.
Disclosure of Invention
In order to solve one of the above technical problems in the prior art, the present disclosure provides a novel coronavirus prototype strain and a characteristic peptide fragment of Delta strain. The present disclosure utilizes the characteristic peptide fragment sequence of novel coronavirus after S protease cleavage, and employs synthetic peptide standard substance (PS) and stable isotope labeled peptide internal standard (SIPS) to complete quantitative analysis, and has high throughput quantitative analysis capability. By adding SIPS, the influence caused by ion inhibition, matrix effect and error in the sample pretreatment process can be greatly reduced, and a more accurate quantitative result can be obtained. Based on the characteristic peptide fragments, the amount of S protein of the novel coronavirus prototype strain and/or Delta strain can be accurately determined.
According to one aspect of the present disclosure, there is provided a polypeptide for quantitative detection of novel coronavirus S proteins. According to an embodiment of the present disclosure, the polypeptide comprises an amino acid sequence as set forth in VGGNYNYLYR (SEQ ID NO: 1). According to an embodiment of the present disclosure, the polypeptide comprises an amino acid sequence as set forth in VGGNYYYR (SEQ ID NO: 2).
According to an embodiment of the disclosure, the polypeptide is unlabeled.
According to an embodiment of the disclosure, the polypeptide is provided with a detectable label. A detectable label refers to any substance that can be detected by mass spectrometry, fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means. Such labels are well known in the art and include, but are not limited to, isotopes (e.g., stable isotopes, such as 2 H、 13 C、 15 N、 17 O、 18 O), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., cy7, alexa 750), acridine esters, magnetic beads (e.g.,) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.), and biotin for binding to the label-modified avidin (e.g., streptavidin) described above.
According to the present disclosureIn an embodiment, the polypeptide is isotopically labeled. According to embodiments of the present disclosure, one or more amino acids in the polypeptide bear an isotopic label. The isotopic label is preferably selected from 2 H、 13 C、 15 N、 17 O、 18 O and any combination thereof.
According to an embodiment of the present disclosure, one or more atoms in valine of the polypeptide are replaced with the corresponding isotope. According to a specific embodiment, valine (V) at position 1 comprising a polypeptide having an amino acid sequence as set forth in VGGNYNYLYR (SEQ ID NO: 1) is isotopically labeled. According to a specific embodiment, valine (V) at position 1 comprising a polypeptide having an amino acid sequence as shown in VGGNYYYR (SEQ ID NO: 2) is isotopically labeled. For example, the C atom on valine is replaced by 13 C, N atoms being replaced by 15 The N, H atoms being replaced by 2 H, O atoms being replaced by 17 O or 18 O。
According to another aspect, a kit for quantitative detection of novel coronavirus S proteins is provided.
According to an embodiment of the disclosure, the kit comprises a first polypeptide comprising an amino acid sequence as set forth in VGGNYNYLYR (SEQ ID NO.: 1). According to some embodiments, the first polypeptide may carry a detectable label.
According to an embodiment of the present disclosure, the kit comprises a second polypeptide comprising an amino acid sequence as set forth in VGGNYYYR (SEQ ID NO.: 2). According to some embodiments, the second polypeptide may carry a detectable label.
According to an embodiment of the present disclosure, the kit comprises: a first polypeptide comprising an amino acid sequence as set forth in VGGNYNYLYR (SEQ ID No.: 1); and a second polypeptide comprising an amino acid sequence as set forth in VGGNYYYRR (SEQ ID NO.: 2). According to some embodiments, the first polypeptide may carry a detectable label. According to some embodiments, the second polypeptide may carry a detectable label.
According to an embodiment of the disclosure, the first polypeptide and/or the second polypeptideOne or more amino acids in the second polypeptide bear an isotopic label. According to a specific embodiment, the isotopic label may be selected from 2 H、 13 C、 15 N、 17 O、 18 O and any combination thereof.
According to an embodiment of the present disclosure, one or more atoms in valine of the first polypeptide and/or the second polypeptide are replaced by corresponding isotopes.
According to an embodiment of the present disclosure, the first polypeptide is used to determine the amount of novel coronavirus prototype strain S protein. According to embodiments of the present disclosure, the novel prototype strain of coronavirus is also known as SARS-CoV-2 or 2019-nCoV.
According to an embodiment of the present disclosure, the second polypeptide is used to determine the amount of novel coronavirus Delta strain S protein. The novel coronavirus Delta strain is named novel coronavirus Delta strain with the number of B.1.617.2, which is the earliest virus variant found in India in month 10 of 2020.
According to an embodiment of the present disclosure, the kit further comprises one or more of a denaturing agent, a reducing agent, an alkylating agent, and a protease.
According to a specific embodiment, the denaturing agent may be selected from urea, sodium dodecyl sulfonate, sodium octane sulfonate or rapidestrsf solution.
According to a specific embodiment, the reducing agent may be selected from dithiothreitol, mercaptoethanol or tris (2-carboxyethyl) phosphine.
According to a specific embodiment, the alkylating agent is selected from iodoacetamides.
According to a specific embodiment, the protease is selected from trypsin.
According to yet another aspect, a method for quantitatively detecting novel coronavirus S protein is provided. The method may include: digesting a sample to be detected by using protease to obtain a digestion product; and determining the amount of the first polypeptide and/or the second polypeptide in the digestion product using high performance liquid chromatography and mass spectrometry.
According to an embodiment of the present disclosure, the first polypeptide comprises an amino acid sequence as set forth in VGGNYNYLYR (SEQ ID NO.: 1). According to an embodiment of the present disclosure, the second polypeptide comprises an amino acid sequence as set forth in VGGNYYYR (SEQ ID NO.: 2).
According to an embodiment of the present disclosure, the method further comprises: in the digestion product, the first polypeptide with a detectable label is added as a first internal standard and/or the second polypeptide with a detectable label is added as a second internal standard. According to a specific embodiment, a standard curve may be drawn based on the amount of the first internal standard for calculating the amount of the first polypeptide, whereby the amount of S protein of the novel coronavirus prototype strain may be calculated. According to a specific embodiment, a standard curve may be drawn based on the amount of the second internal standard for calculating the amount of the second polypeptide, whereby the amount of S protein of the novel coronavirus Delta strain may be calculated.
According to embodiments of the present disclosure, one or more amino acids in the first internal standard and/or the second internal standard are isotopically labeled. According to a specific embodiment, the isotopic label may be selected from 2 H、 13 C、 15 N、 17 O、 18 O and any combination thereof. According to a specific embodiment, the first internal standard may be { Val (13C 5, 15N) } GGNYNYLYR. According to a specific embodiment, the second internal standard may be { Val (13C 5, 15N) } ggnynyyr.
According to an embodiment of the disclosure, the protease is selected from trypsin.
According to an embodiment of the disclosure, the monitoring mode of the mass spectrum is a Parallel Reaction Monitoring (PRM) mode.
As the most potential protein quantification tool, high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) has the characteristics of high sensitivity and high throughput. Common modes for protein quantification using high resolution mass spectrometry include an ion flow chromatography peak (SIM) mode based on primary mass spectrometry cleaved peptide fragment ions, and a Parallel Reaction Monitoring (PRM) mode based on secondary mass spectrometry fragment ions. In both modes, the actual amount of protein is typically represented by the PRM mode chromatographic peak area of the cleaved peptide fragment. The high-resolution mass spectrum utilizes the peptide sequence specific to a protein to finish quantitative analysis, can analyze several to tens of proteins at the same time in one operation, has high-flux quantitative analysis capability, and has wide application prospect in the field of complex protein mixture quantification. In contrast to the affinity method, PRM is not limited by the type and specificity of the protein mixture.
According to a specific embodiment, the sample to be tested is selected from the group consisting of inactivated novel coronavirus 2019-nCoV strain vaccine. According to a specific embodiment, the sample to be tested is selected from the group consisting of inactivated novel coronavirus Delta strain vaccines. According to a specific embodiment, the sample to be tested is selected from inactivated novel coronavirus 2019-nCoV strain + Delta strain vaccines, i.e. comprises both inactivated novel coronavirus 2019-nCoV strain virus and inactivated Delta strain virus.
According to embodiments of the present disclosure, the method for quantitatively detecting novel coronavirus S protein may include the steps of:
(1) Performing enzymolysis pretreatment on a novel coronavirus inactivated vaccine 2019-nCoV strain and/or a Delta strain to obtain an enzymolysis solution;
(2) And (3) performing high performance liquid chromatography-high resolution mass spectrometry detection on the enzymolysis liquid obtained in the step (1), and obtaining the content of S protein of the 2019-nCoV strain and/or the Delta strain in the enzymolysis liquid according to the chromatographic peak area and the standard working curve of the characteristic peptide section of the S protein of the 2019-nCoV strain and/or the Delta strain, wherein the content of S protein of the 2019-nCoV strain and/or the Delta strain in the novel coronavirus inactivated vaccine is obtained through the volume conversion of the enzymolysis liquid and the volume of the novel coronavirus inactivated vaccine 2019-nCoV strain and/or the Delta strain.
According to a specific embodiment, the step (1) may include: taking 500 mu L of novel coronavirus inactivated vaccine, centrifuging at 12000rpm for 10 minutes, discarding supernatant, adding 100 mu L of protein denaturant solution into residues, uniformly mixing, placing into a dry constant temperature metal bath, and heating at 50-90 ℃ for 15 minutes; adding 10 mu L of disulfide bond breaking reagent solution, uniformly mixing, and reacting for 60 minutes at 50-90 ℃; after cooling to room temperature, 10 mu L of alkylating reagent solution is added and mixed uniformly, and the mixture is reacted for 60 minutes at room temperature in a dark place; adding 500 mu L of ammonium bicarbonate solution, 100 mu L of internal standard solution and 10 mu L of trypsin solution, uniformly mixing, reacting for 10-20 hours at 37, adding 5 mu L of acid solution, uniformly mixing, and fixing the volume to 1mL; centrifuging at 12000rpm for 10min, and collecting supernatant, namely enzymolysis solution.
According to a specific embodiment, the high performance liquid chromatography employs an octyl silane bonded silica gel column. According to a specific embodiment, the high performance liquid chromatography employs an octadecylsilane chemically bonded silica column. According to a specific embodiment, the column temperature of the chromatographic column is 50-90 ℃.
According to specific embodiments, the conditions of the high performance liquid chromatography may include: adopting an octyl silane bonded silica gel chromatographic column or an octadecyl silane bonded silica gel chromatographic column; the column temperature is 50-90 ℃; mobile phase A is an aqueous solution containing an ion exchanger; mobile phase B has two forms: a mixed solution of an ion exchanger and an organic solvent or a mixed solution of an ion exchanger, an organic solvent and water; gradient is 0-10 min,5% -32% mobile phase B; 10-10.1 min, 32-90% of mobile phase B; 10.1-12 min,90% mobile phase B; 12-12.1 min, 90-5% of mobile phase B; 12.1-17 min,5% mobile phase B; flow rate: 0.2-0.5 mL/min.
According to a specific embodiment, the conditions for high resolution mass spectrometry detection may include: ESI ion source, positive ion mode; a high resolution mass spectrometer; ion source temperature: 250-350 ℃; auxiliary air flow rate: 5-15 arb; shell air flow rate: 25-45 arb; ion transport capillary: 250-350 ℃; interface voltage: 3.0-4.5 kV and collision energy of 20-40.
According to a specific embodiment, for the first polypeptide, product ion 1119.523 is used as secondary ion for quantification. According to a specific embodiment, for the second polypeptide, product ion 843.375 is used as secondary ion for quantification. According to a specific embodiment, for the first internal standard, product ion 1119.523 is used as secondary ion for quantification. According to a specific embodiment, for the second polypeptide, product ion 843.375 is used as secondary ion for quantification.
According to a specific embodiment, the volume scaling may be performed according to the following formula:
C=C measuring ×V 2 /V 1
Wherein V is 1 Refers to the volume of the enzymolysis liquid, V 2 Volume of liquid to be measured after specified volume, C Measuring The content of the component to be measured in the liquid to be measured is obtained by a standard working curve, and C refers to the content of the component to be measured in the sample to be measured.
According to a specific embodiment, the concentration of the trypsin solution may be 0.2-1. Mu.g/. Mu.L, which may be obtained by dissolving 100. Mu.g trypsin using 100-500. Mu.L of 50mmol/L ammonium bicarbonate solution.
According to a specific embodiment, the ion exchanger in the chromatographic conditions is an acid or a salt, or a mixture of acid and salt, which is also suitable for mass spectrometry. For example, the salt may be selected from ammonium formate or ammonium acetate and the acid may be selected from formic acid or acetic acid.
According to a specific embodiment, in the mobile phase a, the ion exchanger is selected from salts, and the concentration of the aqueous solution of the ion exchanger is 0 to 20mmol/L.
According to a specific embodiment, in the mobile phase A, when the ion exchanger selects acid, the volume ratio of the acid to water is (0-10) to (100-1000).
According to a specific embodiment, in the mobile phase B, when the ion exchanger is an acid, the volume ratio of the ion exchanger, the organic solvent, and the water is (1-2): (200-1000): (0-1000).
According to a specific embodiment, in the mobile phase B, when the ion exchanger is a salt, the salt is dissolved in water to form a brine solution, an organic solvent: the volume ratio of the saline solution is 100-60: 0 to 40, wherein the concentration of the salt solution is 0 to 40mmol/L.
According to specific embodiments, the organic solvent in mobile phase B may include methanol, acetonitrile.
According to a specific embodiment, the concentration of the denaturing agent is 0.05% to 0.2%.
According to a specific embodiment, the acidic solution is selected from one or more of formic acid, acetic acid, trifluoroacetic acid in water. The volume ratio of formic acid, acetic acid, trifluoroacetic acid and water is 100-1:0-100.
According to a specific embodiment, the concentration of the reducing agent may be 50mmol/L to 1mol/L.
According to a specific embodiment, the concentration of the ammonium bicarbonate solution in the sample pretreatment method can be 10-500 mmol/L. According to a specific embodiment, the concentration of the ammonium bicarbonate solution in the sample pretreatment method can be 50-100 mmol/L.
According to a specific embodiment, the concentration of the alkylating agent may be 100mmol/L to 2mol/L. According to a specific embodiment, the concentration of the alkylating agent may be 1mol/L.
According to a specific embodiment, the concentration of the first internal standard may be 200 to 350ng/mL. According to a specific embodiment, the concentration of the second internal standard may be 30 to 40ng/mL.
The invention discloses characteristic polypeptides of S proteins of novel coronavirus inactivated vaccines 2019-nCoV strains and Delta strains, and quantitative determination of the S proteins of the novel coronavirus inactivated vaccines 2019-nCoV strains and/or Delta strains by adopting the characteristic polypeptides. The method adopts a high performance liquid chromatography-high resolution mass spectrometry combined method, and establishes a quantitative method of S protein of different strains in a novel coronavirus inactivated vaccine (2019-nCoV strain, delta strain or 2019-nCoV strain+Delta strain) with high flux, high selectivity and high sensitivity. The method screens out the differential peptide segments which can be used for quantitative analysis of the S protein of the novel coronavirus inactivated vaccine from the complex vaccine matrix for the first time, and can realize the quantification of the S protein of different strains in the novel coronavirus inactivated vaccine (2019-nCoV strain, delta strain or 2019-nCoV strain+Delta strain) in different batches.
The method disclosed by the invention not only can realize the content measurement of the active proteins of different strains of the novel coronavirus inactivated vaccine, but also can be used for comparing the content of the active proteins of different batches of bivalent or multivalent vaccines and evaluating the batch repeatability of products, and can provide a reference for the establishment of related product quality control standards. The present disclosure screens out characteristic peptide fragments of S proteins of novel coronavirus inactivated vaccine 2019-nCoV strain and Delta strain. The screened characteristic peptide has high selectivity and matrix interference resistance, and can be used for quantitative research of active proteins in novel coronavirus inactivated vaccines.
In addition, the method can realize simultaneous content measurement of active proteins of different strains of the novel coronavirus inactivated vaccine by one enzymolysis, one sample injection and liquid phase separation and mass spectrum detection in a very short time (for example, 17 minutes). Moreover, the methods of the present disclosure enable consistent, reproducible, high throughput, accurate quantitative analysis. Compared with the problem that original antigen detection systems cannot respectively and accurately quantify prototype strains and variant strains in bivalent or multivalent vaccines, the method disclosed by the invention is not limited by the types and the specificities of protein mixtures, and can obtain accurate relative quantitative results.
Drawings
FIG. 1 shows typical chromatograms of a polypeptide standard and an internal standard polypeptide standard. In FIG. 1, the chromatograms of polypeptide VGGNYNYLYR, polypeptide { Val (13C 5, 15N) } GGNYNYLYR, polypeptide VGGNYNYR, polypeptide { Val (13C 5, 15N) } GGNYYNYR are shown from top to bottom.
Figure 2 shows a typical secondary mass spectrum of a prototype strain characteristic polypeptide VGGNYNYLYR standard substance.
FIG. 3 shows a typical secondary mass spectrum of a standard substance of a prototype strain internal standard polypeptide { Val (13C 5, 15N) } GGNYNYLYR.
FIG. 4 shows a typical secondary mass spectrum of the Delta strain characteristic polypeptide VGGNYYYNR standard substance.
FIG. 5 shows a typical secondary mass spectrum of the Delta strain internal standard polypeptide { Val (13C 5, 15N) } GGNYNYYNR standard substance.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
Definition of the definition
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.
During replication of the virus, the nucleic acid sequence is sometimes mutated. A virus strain having one or more new mutations may be referred to as a "variant" of the original virus strain. The longer the virus is propagated, the greater the probability of mutation. These mutations may result in changes in viral characteristics, such as altered ability to adapt to the environment, transmissibility or severity, as compared to the original strain. This process of altering and selecting for successful variation is known as virus evolution.
Examples and figures are provided below to aid in the understanding of the invention. It is to be understood that these examples and drawings are for illustrative purposes only and are not to be construed as limiting the invention in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention.
The multivalent novel coronavirus inactivated vaccine (prototype strain+Delta strain) comprises a novel coronavirus prototype strain and a Delta strain, or other strains are added on the basis of the two strains. The preparation method comprises the following steps: the virus of different strains is inoculated with African green monkey kidney cells (Vero cells for short) respectively, and is prepared by virus culture, virus liquid harvesting, virus inactivation, concentration, purification and aluminum hydroxide adsorption. Is used for preventing diseases caused by new coronavirus infection.
Sources of instruments, reagents and materials used in the examples
1. Main experimental instrument: high performance liquid chromatographs (including high pressure binary pumps, deaerators, autosamplers, column incubators) were purchased from sameiser technologies; high resolution mass spectrometers were purchased from sammer feishier technologies.
2. Experimental reagent: rapidest is available from waters science and technology limited; formic acid, acetic acid, trifluoroacetic acid, ammonium bicarbonate, dithiothreitol, tris (2-carboxyethyl) phosphine, urea, sodium dodecyl sulfonate, sodium octane sulfonate, and iodoacetamide were purchased from sigma aldrich (Shanghai) trade company; methanol and acetonitrile were purchased from sammer feishier technologies; trypsin was purchased from prasugrel biotechnology limited (beijing).
3. Novel coronavirus inactivated vaccines (prototype strain + Delta strain, bivalent vaccine) are provided by kexing vitamin technologies limited.
4. The synthetic Polypeptide Standard (PS) and the stable isotope labeled polypeptide internal standard (SIPS) were synthesized by Kirschner Biotech Co., ltd.
Examples
EXAMPLE 1 determination of quantitative peptide fragments of S proteins of novel coronavirus prototype strains and Delta strains and quantitative ion pair
The original strain S protein and the Delta strain S protein are subjected to trypsin digestion to form 10 differential peptide fragments, and the differential peptide fragments are screened out to be combined into an original strain peptide fragment VGGNYNYLYR (SEQ ID NO: 1) and a Delta strain peptide fragment VGGNYNYR (SEQ ID NO: 2).
Prototype plant characteristic polypeptide VGGNYNYLYR (SEQ ID NO: 1), prototype plant internal standard polypeptide { Val (13C 5, 15N) } GGNYNYLYR, delta plant characteristic polypeptide VGGNYYYNR (SEQ ID NO: 2) and Delta plant internal standard polypeptide { Val (13C 5, 15N) } GGNYYNR were synthesized.
The high performance liquid chromatography tandem mass spectrometry detection analysis is carried out according to the following steps.
1. Detection conditions
Chromatographic column: waters ACQUITY UPLC Peptide BEH C18 chromatography column. Mobile phase: a-formic acid: water (1:1000, v/v); b-formic acid: acetonitrile (1:1000, v/v); gradient: 0-10 min, 5-32% B;10 to 10.1min,32 to 90 percent of B; 10.1-12 min,90% B; 12-12.1 min, 90-5% B;12.1 to 17 minutes, 5 percent of B; flow rate: 0.4 mL/min; column temperature is 60 ℃; sample injection volume: 10 mu L.
Mass spectrometry conditions: ion source: esi+ mode; mass spectrometer: a high resolution mass spectrometer; ion source temperature: 320 ℃; auxiliary air flow rate: 10arb; shell air flow rate: 40arb; ion transport capillary: 350 ℃; interface voltage: 4kV. The mass spectrometer detector detection mode is a Parallel Reaction Monitoring (PRM) mode.
The mass spectrum detection parameters are shown in table 1.
TABLE 1 PRM detection parameters of Mass Spectrometry
The chromatogram of the 4 characteristic peptide fragments shown in Table 1 is shown in FIG. 1, and the mass spectrum results are shown in FIGS. 2 to 5, respectively.
From the results of fig. 2, it can be seen that 1119.523 fragment ions, which are higher in response and higher in mass-to-charge ratio, can be used as secondary ions for quantification. From the results of fig. 3, it can be seen that 1119.523 fragment ions, which are higher in response and higher in mass-to-charge ratio, can be used as secondary ions for quantification. From the results of fig. 4, it can be seen that 843.375 fragment ions, which are higher in response and higher in mass-to-charge ratio, can be used as secondary ions for quantification. From the results of fig. 5, it can be seen that 843.375 fragment ions, which are higher in response and higher in mass-to-charge ratio, can be used as secondary ions for quantification.
Example 2 anti-matrix interference test
Prototype strain: taking 100 mu L of S protein stock solution of a prototype strain with the concentration of 50 mu g/mL, placing the stock solution in a low adsorption centrifuge tube, centrifuging at 12000rpm for 10 minutes, discarding supernatant, adding 100 mu L of internal standard solution and 100 mu L of protein denaturant solution into residues, uniformly mixing, placing the residues in a dry constant-temperature metal bath, and heating at 50-90 ℃ for 15 minutes; adding 10 mu L of disulfide bond breaking reagent solution, uniformly mixing, and reacting for 60 minutes at 50-90 ℃; after cooling to room temperature, 10 mu L of alkylating reagent solution is added and mixed uniformly, and the mixture is reacted for 60 minutes at room temperature in a dark place; adding 500 mu L of ammonium bicarbonate solution and 10 mu L of trypsin solution, uniformly mixing, reacting for 10-20 hours at 37 ℃, adding 5 mu L of acid solution, uniformly mixing, and fixing the volume to 1mL; centrifuging at 12000rpm for 10min, and collecting supernatant, namely enzymolysis solution. The enzymatic hydrolysate was subjected to liquid chromatography and PRM mass spectrometry according to the conditions determined in example 1. If the obtained PRM mass spectrum has no other response (i.e., the response intensity is 0), or has a response intensity value but is only baseline noise (i.e., no chromatographic peak is detected), the prototype strain characteristic peptide fragment is the prototype strain characteristic peptide fragment, and cannot be obtained from other matrix proteolysis, i.e., the peptide fragment has specificity and matrix interference resistance.
In the extracted ion flow chromatogram, a chromatographic peak with the same retention time as the polypeptide VGGNYNYLYR standard substance appears in the prototype strain chromatogram, and a chromatographic peak with the same retention time as the polypeptide VGGNYYYNR standard substance does not appear.
Delta strain: 100 mu L of S protein stock solution of Delta strain with the concentration of 50 mu g/mL is taken and placed in a low adsorption centrifuge tube, and enzymolysis, liquid chromatography and PRM mass spectrometry are carried out according to the method. If the obtained PRM mass spectrogram has no other response (i.e. the response intensity is 0), or has a response intensity value but is only baseline noise (i.e. no chromatographic peak is detected), the characteristic peptide fragment of the Delta strain is the characteristic peptide fragment of the Delta strain, and cannot be obtained from other matrix proteolysis, namely the peptide fragment has specificity and matrix interference resistance.
In the extracted ion flow chromatogram, a chromatographic peak with the same retention time as the polypeptide VGGNYYYRR standard substance appears in the Delta chromatogram, and a chromatographic peak with the same retention time as the polypeptide VGGNYNYLYR standard substance does not appear.
EXAMPLE 3 Linear analysis and quantitative Limit
1. Linearity of
(1) Preparation of Peptide Standard (PS) stock solution: the solid of the polypeptide standard substance (PS) is taken and dissolved into standard stock solution with the concentration of 1 mug/mL by 50mmol/L ammonium bicarbonate solution respectively.
(2) Preparing stable isotope labeled peptide internal standard stock solution: the solid of the internal standard substance (SIPS) of the stable isotope labeled polypeptide is respectively dissolved into standard stock solution with the concentration of 1 mug/mL by 50mmol/L ammonium bicarbonate solution.
(3) Standard series solution configuration: the standard stock solutions were measured in appropriate amounts to prepare standard series solutions, and the results are shown in Table 2.
Obtaining a standard curve equation: y=ax+b; r is (r) 2
Wherein:
the ratio of the concentration of the X-protein characteristic polypeptide to the concentration of the isotopically labeled characteristic polypeptide;
the ratio of the peak area of the Y-protein characteristic polypeptide to the peak area of the isotope internal standard characteristic polypeptide;
a-constant;
b-constant;
r 2 -a correlation coefficient.
2. Quantitative Limit (LOQ)
The results of the method, which were obtained by using each characteristic polypeptide as a monitoring target and using the corresponding protein concentration as the lower limit of the quantification of the method, namely LOQ (μg/mL), when the response of the peptide fragment was about 10 times of noise, are shown in Table 2.
Example 4 repeatability assay
1. Repeatability of enzymolysis
10 samples of the bivalent seedlings of the prototype strain and the Delta strain are taken, and the contents are combined and uniformly mixed. mu.L of the sample solution was centrifuged at 12000rpm for 10 minutes, and the supernatant was discarded. To the residue, 100. Mu.L of the internal standard solution and 100. Mu.L of a 0.1% solution of Rapid were added, and the mixture was stirred well, placed in a dry constant temperature metal bath, and heated at 60℃for 15 minutes. Then, 10. Mu.L of 500mmol/L dithiothreitol solution was added thereto and heated at 60℃for 60 minutes. After cooling to room temperature, the reaction was carried out in a dark place, 10. Mu.L of 1mmol/L iodoacetamide solution was added, and the reaction mixture was left at room temperature for 60 minutes. 500. Mu.L of ammonium bicarbonate solution and 10. Mu.L of trypsin solution are added for enzymolysis for 16 hours at 37 ℃. Add 4. Mu.L trifluoroacetic acid and mix well and leave it at 37℃for 30min. The volume was set to 1mL, centrifuged at 12000rpm for 5min, 10. Mu.L of the supernatant was sampled and analyzed, and the same procedure was repeated 6 times. The enzymolysis liquid obtained in 6 times is respectively sampled, the peak areas of the obtained peptide fragments are subjected to relative standard deviation, and the results are shown in Table 2.
2. Sample injection repeatability
One of the enzymatic hydrolysis repetitive solutions was sampled 5 times continuously, 10. Mu.L each time was sampled, and the peak areas of the obtained peptide fragments were subjected to relative standard deviation, and the results are shown in Table 2.
TABLE 2 protein method repeatability and enzymatic hydrolysis repeatability investigation results
EXAMPLE 5 determination of the S protein content of prototype strain and Delta Strain in novel coronavirus inactivated vaccine (prototype strain+Delta Strain)
Detection conditions: as in example 1.
Standard curve: same as in example 3.
Sample treatment: 10 samples of prototype strain+Delta bivalent seedlings (medium dose) are mixed and evenly mixed, 1000 mu L of sample solution is taken for 12000rpm for centrifugation for 10 minutes, the supernatant is removed, 100 mu L of internal standard solution and 100 mu L of 0.1% RapiGest solution are added into residues and evenly mixed, and the mixture is placed in a dry constant temperature metal bath and heated at 60 ℃ for 15 minutes; 10. Mu.L of 500mmol/L dithiothreitol solution was added and heated at 60℃for 60min; after cooling to room temperature, the mixture was operated in a dark place, 10. Mu.L of 1mmol/L iodoacetamide solution was added, and the mixture was left at room temperature for 60 minutes; adding 500 mu L of ammonium bicarbonate solution, adding 10 mu L of trypsin solution, and carrying out enzymolysis for 14 hours at 37 ℃; adding 4 mu L of trifluoroacetic acid, uniformly mixing, and standing at 37 ℃ for 30min; constant volume to 1mL, centrifuging at 12000rpm for 5min, and taking 10 μl of supernatant for sample injection analysis.
Taking 5 samples of prototype strain +Delta bivalent seedlings (high dosage), mixing the contents, centrifuging at 12000rpm for 10min with 500 μl of sample solution, discarding supernatant, adding 100 μl of internal standard solution and 100 μl of 0.1% RapiGest solution into the residue, mixing, placing in dry constant temperature metal bath, and heating at 60deg.C for 15min; 10. Mu.L of 500mmol/L dithiothreitol solution was added and heated at 60℃for 60min; after cooling to room temperature, the mixture was operated in a dark place, 10. Mu.L of 1mmol/L iodoacetamide solution was added, and the mixture was left at room temperature for 60 minutes; adding 500 mu L of ammonium bicarbonate solution, adding 10 mu L of trypsin solution, and carrying out enzymolysis for 14 hours at 37 ℃; adding 4 mu L of trifluoroacetic acid, uniformly mixing, and standing at 37 ℃ for 30min; the solution was centrifuged at 12000rpm for 5min at 1mL to give 10. Mu.L of supernatant, which was analyzed.
The characteristic polypeptide content in the sample is calculated according to the following formula:
Mt=(X×Ci×V1)/V2
wherein:
the content of the characteristic polypeptide in the Mt-sample, ng/mL;
concentration ratio of characteristic polypeptide to isotopically internal standard characteristic polypeptide in X-sample;
ci-isotope internal standard characteristic polypeptide concentration, ng/mL;
v1-final constant volume of sample, mL;
v2-final measurement of the sample volume of the sample solution, mL.
And after the volume of the obtained sample enzymolysis liquid is fixed, carrying out analysis and detection by using a liquid chromatography tandem high-resolution mass spectrum, and calculating the concentration of S protein in the vaccine to-be-detected samples with the two different contents by using the peak areas of the obtained characteristic peptide fragments.
The results showed that in the prototype strain +Delta bivalent seedling (medium dose) sample, the prototype strain S-containing protein was 24.8ng/ml as polypeptide VGGNYNYLYR and the Delta strain S-containing protein was 1.7ng/ml as polypeptide VGGNYYNYR.
In the prototype strain +Delta bivalent seedling (high dose) sample, the prototype strain-containing S protein is 49.6ng/ml calculated by polypeptide VGGNYNYLYR, and the Delta strain-containing S protein is 3.3ng/ml calculated by polypeptide VGGNYNYR.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.
Sequence listing
<110> vitamin technology Co.Ltd in Beijing Koxing
<120> method for detecting S protein content in novel coronavirus vaccine
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
1 5 10
<210> 2
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Val Gly Gly Asn Tyr Asn Tyr Arg
1 5
Claims (10)
1. A polypeptide for the quantitative detection of a novel coronavirus S protein, characterized in that the polypeptide comprises an amino acid sequence as shown in VGGNYNYLYR (SEQ ID No.: 1) or VGGNYNYR (SEQ ID No.: 2).
2. The polypeptide of claim 1, wherein the polypeptide is unlabeled or carries a detectable label,
preferably, one or more amino acids in the polypeptide bear an isotopic label, preferably selected from the group consisting of 2 H、 13 C、 15 N、 17 O、 18 O and any combination thereof,
preferably, one or more atoms in valine of the polypeptide are replaced by the corresponding isotope.
3. A kit for quantitative detection of novel coronavirus S protein, said kit comprising:
a first polypeptide comprising an amino acid sequence as set forth in VGGNYNYLYR (SEQ ID NO: 1), and/or
A second polypeptide comprising an amino acid sequence as set forth in VGGNYNYR (SEQ ID No.: 2);
preferably, one or more amino acids of the first polypeptide and/or the second polypeptide are provided with a detectable label, preferably an isotopic label, more preferably selected from the group consisting of 2 H、 13 C、 15 N、 17 O、 18 O and any combination thereof,
preferably, one or more atoms in valine of the first polypeptide and/or the second polypeptide are replaced by the corresponding isotopes.
4. The kit of claim 3, wherein the first polypeptide is used to determine the amount of novel coronavirus 2019-nCoV strain S protein and/or the second polypeptide is used to determine the amount of novel coronavirus Delta strain S protein.
5. The kit of claim 3 or 4, further comprising one or more of a denaturing agent, a reducing agent, an alkylating agent, and a protease,
preferably, the denaturant is selected from urea, sodium dodecyl sulfonate, sodium octane sulfonate or rapidestrsf solution;
preferably, the reducing agent is selected from dithiothreitol, mercaptoethanol or tris (2-carboxyethyl) phosphine;
preferably, the alkylating agent is selected from iodoacetamides;
preferably, the protease is selected from trypsin.
6. A method for quantitatively detecting a novel coronavirus S protein, the method comprising:
digesting a sample to be detected by using protease to obtain a digestion product; and
determining the amount of the first polypeptide and/or the second polypeptide in the digestion product using high performance liquid chromatography and mass spectrometry,
wherein the first polypeptide comprises an amino acid sequence as set forth in VGGNYNYLYR (SEQ ID NO: 1) and the second polypeptide comprises an amino acid sequence as set forth in VGGNYYYRR (SEQ ID NO: 2).
7. The method of claim 6, wherein the method further comprises:
adding said first polypeptide with a detectable label as a first internal standard and/or adding said second polypeptide with a detectable label as a second internal standard to said digestion product;
a standard curve is drawn based on the amount of the first internal standard and/or the second internal standard,
preferably, one or more amino acids in the first internal standard and/or the second internal standard are provided with an isotopic label, preferably selected from the group consisting of 2 H、 13 C、 15 N、 17 O、 18 O and any combination thereof,
preferably, the protease is selected from trypsin.
8. The method of claim 6 or 7, wherein the high performance liquid chromatography employs an octyl silane bonded silica gel column or an octadecyl silane bonded silica gel column;
preferably, the column temperature of the chromatographic column is 50-90 ℃.
9. The method according to any one of claims 6 to 8, wherein for the first polypeptide, product ion 1119.523 is used as a secondary ion for quantification; for the second polypeptide, product ion 843.375 was used as a secondary ion for quantification.
10. The method according to any one of claims 7 to 9, wherein the sample to be tested is selected from an inactivated novel coronavirus 2019-nCoV strain vaccine, an inactivated novel coronavirus Delta strain vaccine, or an inactivated novel coronavirus 2019-nCoV strain +delta strain vaccine.
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