CN112684060A - Method for detecting content of novel coronavirus S protein in novel coronavirus inactivated vaccine - Google Patents
Method for detecting content of novel coronavirus S protein in novel coronavirus inactivated vaccine Download PDFInfo
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Abstract
The invention provides a method for detecting the content of a novel coronavirus S protein in a novel coronavirus inactivated vaccine. The detection method utilizes the quantitative peptide fragment (GWIFGTTLDSK) of the invention; the mass-to-charge ratio of the parent ion to the daughter ion of the peptide fragment was 612.8161, and the mass-to-charge ratio of the daughter ion was 244.1080 and/or 216.1080. And (3) utilizing the quantitative peptide segment and the quantitative ion pair, preparing a standard curve by an S protein standard substance through a high performance liquid chromatography tandem mass spectrometry quantitative detection method, carrying out quantitative detection after incubation and enzymolysis on the new coronavirus vaccine to be detected, obtaining the ion peak area of the quantitative peptide segment in the sample, and further calculating to obtain the content of the new coronavirus S protein in the sample. The method has high detection accuracy and high precision, avoids the influence of matrix effect and the like in the vaccine sample on the detection, and can be used for detecting the content of the new coronavirus S protein in intermediate products and finished products of each stage of the new coronavirus inactivated vaccine.
Description
Technical Field
The invention belongs to the technical field of detection of novel coronavirus (SARS-CoV-2) inactivated vaccines, relates to a method for detecting the content of novel coronavirus S protein in the novel coronavirus inactivated vaccines, and particularly relates to a method for quantitatively detecting the content of the novel coronavirus S protein in the novel coronavirus inactivated vaccines by using liquid chromatography tandem mass spectrometry.
Background
The new coronavirus (SARS-CoV-2) epidemic situation discovered in 2019 quickly wraps the world, and brings great loss to the life health of people in various regions and the economic development of society. The diameter of SARS-CoV-2 virus is 60-140 nm, and there are mainly 4 kinds of structural proteins, S (spinous process) protein, N (nucleocapsid) protein, M (membrane) protein and E (envelope) protein. Among them, the S protein is the most important surface membrane protein of the virus, is also an important antigen for causing immune response, and is a key target point for vaccine design. The vaccine is an effective way for sniping and preventing new coronavirus, and becomes the focus of attention of drug research and development all over the world. Inactivated vaccines are the most effective vaccine development approach for new outbreak infectious diseases. The method has the advantages of mature production process, controllable quality standard, wide protection range and the like, can be used for large-scale inoculation, and has international passing standards for judging the safety and effectiveness of the vaccine. The detection of the content of the effective antigen in the inactivated vaccine is particularly critical.
Inactivated vaccines are the most effective vaccine development approach for new outbreak infectious diseases. The method has the advantages of mature production process, controllable quality standard, wide protection range and the like, can be used for large-scale inoculation, and has international passing standards for judging the safety and effectiveness of the vaccine. The detection of the content of the effective antigen in the inactivated vaccine, such as the content determination of S protein and N protein in the new coronavirus, has very important significance for the evaluation of the vaccine.
The traditional method for detecting the content of the novel coronavirus S protein in the inactivated vaccine needs to rely on an immunological analysis method, such as a double-antibody sandwich enzyme-linked immunosorbent assay. The S protein in the novel coronavirus is detected by adopting an enzyme-linked immunosorbent assay technology, and a monoclonal or polyclonal antibody is prepared by utilizing a recombinant expression S protein antigen and performing basic immunization and boosting immunization on a mouse or a rabbit. Coating the purified anti-S protein antibody on a solid phase carrier such as an enzyme-linked reaction plate, sealing the dry plate, adding a product to be detected, performing incubation and plate washing operation, adding an enzyme-labeled anti-S protein antibody to form a labeled immune complex, and removing the unbound enzyme conjugate and other substances by washing. The marker is detected by means of color development or luminescence, and the S protein content of SARS-CoV-2 in the product to be detected can be quantitatively detected by means of a standard curve. The technology for detecting the S protein by using the enzyme-linked method needs a large amount of time to prepare a specific antibody through an animal immunization process, has high requirements on the quality of the antibody, and not only needs extremely high specificity, but also the antibody generally needs high affinity. The enzyme-linked immunoassay is susceptible to the interference of other components in a detection sample in the detection process to influence the detection result, so that the quantification is inaccurate. The ELISA technology has complex experimental operation process, needs 3-5 steps of incubation and washing operation of an ELISA reaction plate, has great influence on the experiment due to artificial factors, and is easy to be influenced by environmental factors in the reaction process, so the stability and the repeatability are poor. Due to the narrow range of ELISA normalcy, 10 is typical2For samples of unknown concentration, pre-compaction is requiredThe test or sample dilution can accurately determine the sample falling within the optimal linear range, and excessive dilution generally causes inaccuracy in sample detection.
In recent years, the liquid chromatography tandem mass spectrometry technology has the advantages of high selectivity, high sensitivity and the like in the aspect of protein detection and analysis, and is very suitable for qualitative and quantitative research of target proteins in complex biological matrixes.
Disclosure of Invention
Based on the prior art to detect the defects of the novel coronavirus S protein in the novel coronavirus inactivated vaccine, the invention aims to provide a quantitative peptide segment for quantitatively detecting the novel coronavirus S protein by liquid chromatography-tandem mass spectrometry; the second purpose of the invention is to provide the application of the quantitative peptide fragment in the quantitative detection of the novel coronavirus S protein by liquid chromatography-tandem mass spectrometry; the third objective of the invention is to provide a method for detecting the content of the novel coronavirus S protein in the novel coronavirus inactivated vaccine, the quantitative peptide fragment and the quantitative ion pair of the S protein quantitative detection of the novel coronavirus obtained by screening are utilized, the method comprises the steps of preparing a standard curve by using an S protein standard substance, carrying out sample treatment and enzymolysis on an intermediate product and a finished product of the new coronavirus to be detected, carrying out high performance liquid chromatography tandem mass spectrometry quantitative detection to obtain an MRM peak area (namely a daughter ion peak area) of a quantitative peptide section in a sample, obtaining the content of the quantitative peptide section of the new coronavirus SARS-CoV-2 in the inactivated vaccine sample to be detected based on the standard curve and a sample injection volume, and dividing the content by the sample injection volume to obtain the concentration of the new coronavirus S protein in the inactivated vaccine sample to be detected.
The purpose of the invention is realized by the following technical scheme:
in one aspect, the invention provides a quantitative peptide segment for quantitative detection of novel coronavirus S protein by liquid chromatography-tandem mass spectrometry, wherein the amino acid sequence of the quantitative peptide segment is as follows:
GWIFGTTLDSK(SEQ ID NO：1)。
the quantitative peptide fragment has the advantages of strong specificity, high response value, strong sequence stability in intermediate products and finished products of different novel coronavirus vaccines and the like.
In the above-mentioned quantitative peptide fragment, preferably, the mass-to-charge ratio of the parent ion to the parent ion in the quantitative ion pair of the quantitative peptide fragment is 612.8161, and the mass-to-charge ratio of the daughter ion is 244.1080 and/or 216.1080.
The quantitative peptide fragment and the quantitative ion pair thereof are applied to the quantitative detection of the novel coronavirus S protein by the liquid chromatography-tandem mass spectrometry, have high detection accuracy and high precision, avoid the influence of virus protein structural change, protein modification, matrix effect and the like on the detection in a novel coronavirus inactivated vaccine sample, and can be used for detecting the content of the novel coronavirus S protein of the sample at each process stage in the novel coronavirus inactivated vaccine.
On the other hand, the invention also provides application of the quantitative peptide fragment in quantitative detection of the novel coronavirus S protein by liquid chromatography-tandem mass spectrometry.
In another aspect, the present invention also provides a method for detecting the content of the novel coronavirus S protein in the novel inactivated coronavirus vaccine, which comprises the following steps:
incubating S protein standard of the recombinant novel coronavirus SARS-CoV-2 and performing enzymolysis treatment; adopting high performance liquid chromatography to connect with triple quadrupole mass spectrometry detection equipment (HPLC-MRM-MS/MS), feeding an S protein standard substance of the recombinant novel coronavirus SARS-CoV-2 after enzymolysis treatment, and drawing a standard curve by taking the content of the standard substance as a horizontal coordinate and taking the peak area of the daughter ion of the quantitative peptide section as a vertical coordinate;
taking an inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected for incubation and carrying out enzymolysis treatment; and (3) carrying out sample injection detection on the supernatant after enzymolysis, obtaining the content of the quantitative peptide segment of the novel coronavirus SARS-CoV-2 in the inactivated vaccine sample to be detected based on a standard curve and a sample injection volume according to the peak area of the daughter ion of the quantitative peptide segment, and dividing the content by the sample injection volume to obtain the concentration of the novel coronavirus S protein in the inactivated vaccine sample to be detected.
In the above detection method, the S protein standard of the recombinant novel coronavirus SARS-CoV-2 is commercially available.
In the above detection method, preferably, the incubation process of the standard S protein of the recombinant novel coronavirus SARS-CoV-2 or the inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected comprises:
adding RapidEST into the S protein standard substance of the recombinant novel coronavirus SARS-CoV-2 or the inactivated vaccine sample to be detected in a medium volume, incubating at 70-90 ℃ for 20-60 min (destroying the virus membrane structure and releasing the effective antigen components), then adding Dithiothreitol (DTT) to a final concentration of 25mmol/L (the final concentration of dithiothreitol), incubating at 65 ℃ for 60min, cooling to room temperature, adding iodoacetamide solution to a final concentration of 50mmol/L (the final concentration of iodoacetamide), and incubating at room temperature for 20-60 min in a dark place.
In the above detection method, preferably, the process of performing enzymolysis after incubation includes:
adding NH into 220 μ L S protein standard of the incubated recombinant novel coronavirus SARS-CoV-2 or incubated inactivated vaccine sample to be detected4HCO3Adding 20 μ L of trypsin solution to 480 μ L of the solution, performing enzymolysis at 37 ℃ overnight, and adding 1 μ L of formic acid to inactivate trypsin after the enzymolysis reaction is completed, thus finishing the enzymolysis reaction.
The invention optimizes the sample treatment and enzymolysis conditions of the novel coronavirus inactivated vaccine, and greatly improves the sample enzymolysis efficiency by optimizing the types and concentrations of surfactants, the alkylation process, the trypsin dosage ratio, the enzymolysis time and the like.
In the above-mentioned detection method, preferably, the conditions of the HPLC detection of the S protein standard of the recombinant novel coronavirus SARS-CoV-2 or the inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected are as follows:
a chromatographic column: a C18 column, preferably ACQUITY UPLC PETIDE BEH C18(2.1mm × 100mm), with a sample amount of 5-10 μ L and a chromatographic column temperature of 60 deg.C;
mobile phase composition: the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is acetonitrile solution containing 0.1% formic acid; the content of the mobile phase B is changed from 0 to 45 percent within 0 to 20min, and gradient elution is carried out, wherein the flow rate is 0.3 mL/min.
In the above-mentioned detection method, preferably, the mass spectrometric detection conditions of the S protein standard of the recombinant novel coronavirus SARS-CoV-2 or the inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected are as follows:
ion source parameters: the flow rate of the atomizer is 3.0L/min, the flow rate of the dryer is 10.0L/min, the interface temperature is 300 ℃, the desolventizing gas temperature is 526 ℃, and the electric spray voltage is 3.0 kV; the DL temperature was 250 ℃.
In the above detection method, preferably, the deviation (v) -20.0 of Q1 Pre of the quantitative ion pair having the parent ion mass-to-charge ratio of 612.8161 and the daughter ion mass-to-charge ratio of 244.1080; CE-20; q3 Pre was biased (v) -17.0.
In the above detection method, preferably, the deviation (v) -15.0 of Q1 Pre of the quantitative ion pair having the parent ion mass-to-charge ratio of 612.8161 and the daughter ion mass-to-charge ratio of 216.1080; CE-24; q3 Pre was biased (v) -15.0.
The invention has the beneficial effects that:
(1) the quantitative peptide fragment has the advantages of strong specificity, high response value, strong sequence stability in intermediate products and finished products of different novel coronavirus vaccines and the like.
(2) The quantitative peptide fragment and the quantitative ion pair thereof are applied to the quantitative detection of the novel coronavirus S protein by the liquid chromatography tandem mass spectrometry, have the characteristics of good detection linear relation, wide linear range, high accuracy, strong specificity, high sensitivity and the like, avoid the influence of virus protein structure change, protein modification, matrix effect and the like in a novel coronavirus inactivated vaccine sample on the detection, and can be used for detecting the content of the novel coronavirus S protein of intermediate products and finished products of various stages of a novel coronavirus inactivated vaccine.
Drawings
FIG. 1 is an ion flow diagram of a quantified peptide fragment GWIFGTTLDSK obtained by screening in example 1 of the present invention.
FIG. 2 is a secondary fragment mass spectrum of the quantitative peptide GWIFGTTLDSK obtained from the screening in example 1 of the present invention.
FIG. 3 is a standard graph of the quantitative peptide GWIFGTTLDSK of the S protein of the inactivated coronavirus vaccine of example 2.
FIG. 4 is the MRM map of the S protein quantitative peptide GWIFGTTLDSK detected by supernatant injection of the novel coronavirus vaccine after enzymolysis in example 2.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
Example 1 the quantitative peptide fragment and quantitative ion pair screening process of the present invention:
performing high-resolution mass spectrometry on the new coronavirus inactivated vaccine product by using a liquid chromatography flight time mass spectrum, and screening characteristic peptide segments and primary parent ions and secondary product ion information thereof, wherein the specific process comprises the following steps:
1. reagents used for screening:
50mmol/L NH4HCO3Solution: 0.04g of NH are weighed4HCO3Adding 10mL of ultrapure water for dissolving.
500mmol/L DTT solution: 0.37mg of DTT was weighed out using 50mmol/L NH4HCO3The solution was made up to 4.0 mL.
1mol/L iodoacetamide solution: 0.37g of iodoacetamide is weighed out, using 50mmol/L NH4HCO3The solution was made up to 4.0 mL.
0.1% rapidest solution: 1mg of RapidEST was weighed out and 1mL of 50mmol/L NH was added4HCO3Dissolving the solution to obtain the product.
Trypsin solution: 20. mu.g of trypsin solution was weighed out in 200. mu.L of 50mmol/L NH4HCO3Obtained in solution.
2. S protein standard of recombined novel coronavirus SARS-CoV-2 and inactivated vaccine sample treatment of novel coronavirus SARS-CoV-2:
taking S protein standard of recombinant novel coronavirus SARS-CoV-2 or novel coronavirus inactivated vaccine sample to be detected, adding 0.1% RapidEST solution in equal volume, and heating at 70 deg.CAnd (3) incubating for 40min, adding 500mmol/L DTT solution to a final concentration of 25mmol/L, incubating for 40min at 65 ℃, cooling to room temperature, adding 1mol/L iodoacetamide solution to a final concentration of 50mmol/L, and reacting for 30min at room temperature in a dark place. Then 50mmol/L NH was added to 220. mu.L of the sample solution4HCO3Adding 20 μ L of trypsin solution into each sample, performing enzymolysis at 37 deg.C overnight, and adding 1 μ L of formic acid to inactivate trypsin after the enzymolysis reaction is completed, thereby finishing the enzymolysis reaction.
3. Mass spectrometric detection and analysis of a sample of a novel coronavirus inactivated vaccine stock solution (supernatant obtained after the above enzymatic hydrolysis):
and detecting by adopting a liquid chromatography time-of-flight mass spectrum.
The liquid chromatography detection operation is as follows:
a chromatographic column: ACQUITY UPLC PEPTIDE BEH C18(2.1 mm. times.100 mm); mobile phase composition: the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is acetonitrile solution containing 0.1% formic acid; the temperature of the chromatographic column is 60 ℃, the sample injection amount of the sample is 10 mu L, and the content of the mobile phase B is changed from 0-45% within 0-70 min for gradient elution, and the flow rate is 0.3 mL/min.
The mass spectrometric detection procedure was as follows:
and (3) analyzing by using a Xevo G2-XS high-resolution mass spectrometer in an MSE mode, wherein the acquisition and analysis software is UNIFI, the mass spectrum analysis range is 50-2000 m/z, the capillary voltage is 3.0KV, the desolventizing air flow rate is 600L/h, and the desolventizing air temperature is 250 ℃.
From the results of mass spectrometry determination of the S protein sequence, 5 characteristic peptide sequences with the strongest detection signals and 14 pairs of ion pair mass spectrometry information (as shown in the following table 1) are obtained through analysis. Through validation and analysis of Uniprot and NCBI databases, the 5 peptide fragment sequences are all characteristic sequences of the S protein.
Table 1:
4. the invention relates to a method for screening quantitative peptide fragments and quantitative ion pairs, which comprises the following steps:
screening strategy: firstly, the peptide fragment should belong to the characteristic sequence of the S protein, and secondly, the quantitative peptide fragment and the corresponding quantitative ion pair thereof have higher response in various samples (standard substance, virus culture solution, intermediate product and finished product), so that the detection aim can be achieved. The inventor creatively utilizes a liquid chromatography tandem mass spectrometry method to detect a novel coronavirus S protein standard product and a virus culture solution, an intermediate product and a finished product of a novel coronavirus inactivated vaccine at each preparation stage (the intermediate product refers to a vaccine stock solution product of a previous step of the finished product, and the finished product refers to the novel coronavirus inactivated vaccine prepared after a certain proportion of vaccine adjuvant and auxiliary materials are added into the intermediate product), and according to the response condition of mass spectrum signals of various samples, a quantitative peptide segment and a quantitative ion pair thereof suitable for detecting the novel coronavirus inactivated vaccine sample S protein are obtained through comprehensive screening. The screening method is different from the traditional method which only depends on mass spectrum scoring data to select and determine quantitative peptide fragments and quantitative ion pairs, but creatively screens the quantitative peptide fragments and the quantitative ion pairs which can realize the product detection of each stage according to the screening strategy through the actual detection conditions of samples at different stages. The screening results are shown in table 2 below, where the detection values of the parent ions and the daughter ions in table 2 are mass-to-charge ratio values, and the detection value of each sample is an MRM peak area (mass spectrum signal value).
Table 2:
note: "- - - - -" indicates no mass spectrum signal.
It can be seen from table 2 that the 612.8161(m/z) >244.1080(m/z) and 612.8161(m/z) >216.1080(m/z) ion pairs of the GWIFGTTLDSK peptide fragment have higher mass spectrum signal values for detecting novel coronavirus S protein standard, new coronavirus inactivated vaccine virus culture fluid, intermediate products and finished products, while the ion pairs of other peptide fragments have the condition of not detecting one or more samples, and the analysis has the following reasons: (1) GWIFGTTLDSK peptide fragments can be stably generated in the enzymolysis treatment process of the sample; (2) the peptide segment is stable in the solution after sample treatment; (3) the 612.8161(m/z) >244.1080(m/z) and 612.8161(m/z) >216.1080(m/z) ion pair mass spectrum corresponding values of the peptide fragment are higher. In conclusion, through screening, only two ion pairs of one peptide fragment can meet all requirements, and the sequence of the quantitative peptide fragment for the S protein of the novel coronavirus inactivated vaccine sample is finally determined to be GWIFGTTLDSK, and the sequence of the quantitative ion pair is 612.8161(m/z) >244.1080(m/z) and/or 612.8161(m/z) >216.1080 (m/z). Fig. 1 is an ion flow diagram of GWIFGTTLDSK peptide fragment, and fig. 2 is a secondary fragment mass spectrum of GWIFGTTLDSK peptide fragment. As can be seen from fig. 2, the two daughter ions of the quantitative ion pair of the present invention can be shown in the graph of the quantitative peptide fragment after collision, which confirms that the quantitative daughter ions of the present invention are fragment ions of the parent ion.
Example 2 method for detecting the content of S protein of novel coronavirus in novel coronavirus inactivated vaccine
This example provides a method for detecting the content of S protein of novel coronavirus in inactivated vaccine of novel coronavirus by using the quantitative peptide fragment and quantitative ion pair combined with high performance liquid chromatography tandem triple quadrupole mass spectrometry detection equipment (HPLC-MRM-MS/MS) of example 1.
The detection conditions of the high performance liquid chromatography involved in the present example were as follows:
a chromatographic column: c18 column, preferably ACQUITY UPLC PETIDE BEH C18(2.1mm × 100mm), with sample amount of 10 μ L and chromatographic column temperature of 60 deg.C;
mobile phase composition: the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is acetonitrile solution containing 0.1% formic acid; the content of the mobile phase B is changed from 0 to 45 percent within 0 to 20min, and gradient elution is carried out, wherein the flow rate is 0.3 mL/min.
The mass spectrometric detection conditions involved in this example are as follows:
ion source parameters: the flow rate of the atomizer is 3.0L/min, the flow rate of the dryer is 10.0L/min, the interface temperature is 300 ℃, the desolventizing gas temperature is 526 ℃, and the electric spray voltage is 3.0 kV; the DL temperature is 250 ℃;
q1 Pre bias (v) -20.0 for ion pair 612.8161(m/z) >244.1080 (m/z); CE-20; q3 Pre-bias (v) -17.0;
q1 Pre bias (v) -15.0 for ion pair 612.8161(m/z) >216.1080 (m/z); CE-24; q3 Pre was biased (v) -15.0.
The specific operation is as follows:
(1) adopting 50mmol/L NH to the S protein standard of the recombinant novel coronavirus SARS-CoV-24HCO3The solution was diluted to 75. mu.g/mL. Adding 0.1% RapidEST, incubating at 70 deg.C for 40min, adding 500mmol/L DTT to final concentration of 25mmol/L, incubating at 65 deg.C for 60min, cooling to room temperature, adding 1mol/L iodoacetamide solution to final concentration of 50mmol/L, and incubating at room temperature in dark place for 30 min. Then 50mmol/L NH was added to 220. mu.L of incubated S protein standard4HCO3The volume is 480 mu L, then 20 mu L of trypsin solution is added, the enzymolysis is carried out overnight at the temperature of 37 ℃, after the enzymolysis reaction is finished, 1 mu L of formic acid is added to inactivate the trypsin, and the enzymolysis reaction is finished.
(2) Adopting high performance liquid chromatography to connect with triple quadrupole mass spectrometry detection equipment in series, injecting sample of different volumes to recombine S protein standard substance of the novel coronavirus SARS-CoV-2, and drawing a standard curve by taking the content of the standard substance as a horizontal coordinate (X) and taking the peak area (Y) of the quantitative peptide fragment ion pair 612.8161>244.1080 sub-ions of the novel coronavirus SARS-CoV-2 screened in the example 1 as a vertical coordinate; the standard curve is shown in fig. 3, and the standard equation is as follows:
Y＝3.17816×106X-10850.1，R2＝0.9999279，R＝0.9999640
(3) taking an inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected for incubation and carrying out enzymolysis treatment, wherein the method specifically comprises the following steps:
adding 0.1% RapidEST into an inactivated vaccine sample to be detected, incubating for 40min at 70 ℃, then adding 500mmol/L DTT to a final concentration of 25mmol/L, incubating for 60min at 65 ℃, adding 1mol/L iodoacetamide solution to a final concentration of 50mmol/L after cooling to room temperature, and incubating for 30min at room temperature in a dark place. Is connected withAdding 50mmol/L NH into 220 mu L incubated inactivated vaccine sample to be detected4HCO3The volume is 480 mu L, then 20 mu L of trypsin solution is added, the enzymolysis is carried out overnight at the temperature of 37 ℃, after the enzymolysis reaction is finished, 1 mu L of formic acid is added to inactivate the trypsin, and the enzymolysis reaction is finished.
(4) And (3) carrying out sample injection detection on the supernatant after enzymolysis, wherein the sample injection amount is 5 mu L, obtaining the content of the quantitative peptide segment of the novel coronavirus SARS-CoV-2 in the inactivated vaccine sample to be detected based on a standard curve and a sample injection volume according to the peak area (shown as 01 batches of samples in figure 4) of 612.8161(m/z) >244.1080(m/z) of the quantitative peptide segment of the novel coronavirus SARS-CoV-2, and dividing by the sample injection volume to obtain the concentration of the novel coronavirus S protein in the inactivated vaccine sample to be detected, wherein the specific detection result is shown in the following table 3.
Table 3:
example 3 method for verifying the content of the S protein of the novel coronavirus in the novel coronavirus inactivated vaccine
In this example, the quantitative peptide fragment and the quantitative ion pair of example 2 are combined with a high performance liquid chromatography tandem triple quadrupole mass spectrometry detection device to perform a methodological verification process on a method for detecting the content of the novel coronavirus S protein in the novel coronavirus inactivated vaccine.
The detection conditions of the high performance liquid chromatography involved in the present example were as follows:
a chromatographic column: c18 column, preferably ACQUITY UPLC PETIDE BEH C18(2.1mm × 100mm), with sample amount of 10 μ L and chromatographic column temperature of 60 deg.C;
mobile phase composition: the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is acetonitrile solution containing 0.1% formic acid; the content of the mobile phase B is changed from 0 to 45 percent within 0 to 20min, and gradient elution is carried out, wherein the flow rate is 0.3 mL/min.
The mass spectrometric detection conditions involved in this example are as follows:
ion source parameters: the flow rate of the atomizer is 3.0L/min, the flow rate of the dryer is 10.0L/min, the interface temperature is 300 ℃, the desolventizing gas temperature is 526 ℃, and the electric spray voltage is 3.0 kV; the DL temperature is 250 ℃;
q1 Pre bias (v) -20.0 for ion pair 612.8161(m/z) >244.1080 (m/z); CE-20; q3 Pre-bias (v) -17.0; q1 Pre bias (v) -15.0 for ion pair 612.8161(m/z) >216.1080 (m/z); CE-24; q3 Pre was biased (v) -15.0.
The specific operation is as follows:
(1) linearity: referring to fig. 3, in the standard curve obtained by using the quantitative peptide fragment and the quantitative ion pair of the present invention, the peak area (Y) of the quantitative peptide fragment daughter ion is linearly related to the content (X), and the correlation coefficient is as high as 0.9999279, which has a very good linear relationship.
(2) Quantification limit and detection limit: the concentration when the signal to noise ratio of the quantitative peptide fragment ion detection is more than or equal to 10(S/N is more than or equal to 10) is taken as the quantitative limit; the concentration of quantitive ion with signal-to-noise ratio greater than or equal to 3(S/N is greater than or equal to 3) is used as the detection Limit (LODs). The detection limit and the quantification limit of the measured peptide fragment were 0.0014. mu.g and 0.003. mu.g, respectively. The method is high in sensitivity and can meet the requirement of S protein detection.
(3) Precision: and (4) repeatedly carrying out sample injection and determination for 5 times of enzyme digestion treatment by the method, and calculating the relative standard deviation of the peak area of the daughter ion. The results are shown in Table 4 below.
Table 4:
as can be seen from Table 4, the relative standard deviation (RSD%) of the peak area of the measured peptide fragment daughter ion was about 2.1%, indicating that the method was accurate.
(4) Accuracy: and adding an S protein standard substance into the blank matrix of the stock solution of the new crown inactivated vaccine to be detected, detecting by the sample processing method, measuring the concentration of the added standard sample by using a standard curve, and comparing the measured value with the theoretical value to calculate the added standard recovery rate. The results are shown in Table 5.
Table 5:
as can be seen from Table 5, the average recovery of the measured peptide fragments was 94.2%, and the accuracy of the method was good.
Sequence listing
<110> limited liability company of Beijing institute of biological products
<120> method for detecting content of novel coronavirus S protein in novel coronavirus inactivated vaccine
<130> GAI20CN6436
<160> 5
<170> PatentIn version 3.5
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<212> PRT
<213> SARS-CoV-2
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Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys
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<213> SARS-CoV-2
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Val Val Val Leu Ser Pro Glu Leu Leu His Ala Pro Ala Thr Val Cys
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Gly Pro Lys
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<213> SARS-CoV-2
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Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
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Val Thr Leu Ala Asp Ala Gly Phe Ile Lys
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<212> PRT
<213> SARS-CoV-2
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Phe Leu Pro Phe Gln Gln Phe Gly Arg
1 5
Claims (10)
1. A quantitative peptide segment for quantitative detection of novel coronavirus S protein by liquid chromatography-tandem mass spectrometry has the following amino acid sequence:
GWIFGTTLDSK(SEQ ID NO：1)。
2. the quantitative peptide fragment of claim 1, wherein the mass-to-charge ratio of the parent ion to the parent ion of the quantitative ion pair of the quantitative peptide fragment is 612.8161, and the mass-to-charge ratio of the daughter ion is 244.1080 and/or 216.1080.
3. Use of the quantitative peptide fragment of claim 1 or 2 for the quantitative detection of novel coronavirus S protein by liquid chromatography tandem mass spectrometry.
4. A method for detecting the content of a novel coronavirus S protein in a novel coronavirus inactivated vaccine comprises the following steps:
incubating S protein standard of the recombinant novel coronavirus SARS-CoV-2 and performing enzymolysis treatment; adopting high performance liquid chromatography to connect with triple quadrupole mass spectrometry detection equipment in series, sampling S protein standard substance of the recombined novel coronavirus SARS-CoV-2 after enzymolysis, and drawing a standard curve by taking the content of the standard substance as a horizontal coordinate and taking the peak area of the sub-ion of the quantitative peptide segment of claim 1 or 2 as a vertical coordinate;
taking an inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected for incubation and carrying out enzymolysis treatment; and (3) carrying out sample injection detection on the supernatant after enzymolysis, obtaining the content of the quantitative peptide segment of the novel coronavirus SARS-CoV-2 in the inactivated vaccine sample to be detected based on a standard curve and a sample injection volume according to the peak area of the daughter ion of the quantitative peptide segment, and dividing the content by the sample injection volume to obtain the concentration of the novel coronavirus S protein in the inactivated vaccine sample to be detected.
5. The detection method according to claim 4, wherein the incubation of the S protein standard of the recombinant novel coronavirus SARS-CoV-2 or the inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected comprises:
adding RapidEST into the S protein standard substance of the recombinant novel coronavirus SARS-CoV-2 or the inactivated vaccine sample to be detected in a medium volume, incubating at 70-90 ℃ for 20-60 min, then adding dithiothreitol to a final concentration of 25mmol/L, incubating at 65 ℃ for 60min, cooling to room temperature, adding iodoacetamide solution to a final concentration of 50mmol/L, and incubating at room temperature in a dark place for 20-60 min.
6. The detection method according to claim 4 or 5, wherein the incubation followed by the enzymatic treatment comprises:
adding NH into 220 μ L S protein standard of the incubated recombinant novel coronavirus SARS-CoV-2 or incubated inactivated vaccine sample to be detected4HCO3Adding 20 μ L of trypsin solution to 480 μ L of the solution, performing enzymolysis at 37 ℃ overnight, and adding 1 μ L of formic acid to inactivate trypsin after the enzymolysis reaction is completed, thus finishing the enzymolysis reaction.
7. The method according to claim 4, wherein the conditions for detecting the S protein standard of the recombinant novel coronavirus SARS-CoV-2 or the inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected by high performance liquid chromatography are as follows:
a chromatographic column: a C18 column, preferably ACQUITY UPLC PETIDE BEH C18(2.1mm × 100mm), with a sample amount of 5-10 μ L and a chromatographic column temperature of 60 deg.C;
mobile phase composition: the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is acetonitrile solution containing 0.1% formic acid; the content of the mobile phase B is changed from 0 to 45 percent within 0 to 20min, and gradient elution is carried out, wherein the flow rate is 0.3 mL/min.
8. The method according to claim 4, wherein the mass spectrometric detection conditions of the S protein standard of the recombinant novel coronavirus SARS-CoV-2 or the inactivated vaccine sample of the novel coronavirus SARS-CoV-2 to be detected are as follows:
ion source parameters: the flow rate of the atomizer is 3.0L/min, the flow rate of the dryer is 10.0L/min, the interface temperature is 300 ℃, the desolventizing gas temperature is 526 ℃, and the electric spray voltage is 3.0 kV; the DL temperature was 250 ℃.
9. The detection method according to claim 4 or 8, wherein the deviation (v) -20.0 of Q1 Pre of a quantitative ion pair having a parent ion mass-to-charge ratio of 612.8161 and a child ion mass-to-charge ratio of 244.1080; CE-20; q3 Pre was biased (v) -17.0.
10. The detection method according to claim 4 or 8, wherein the deviation (v) -15.0 of Q1 Pre of a quantitative ion pair having a parent ion mass-to-charge ratio of 612.8161 and a child ion mass-to-charge ratio of 216.1080; CE-24; q3 Pre was biased (v) -15.0.
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