CN112763628A - Method for detecting content of N protein of novel coronavirus in novel coronavirus inactivated vaccine - Google Patents
Method for detecting content of N protein of novel coronavirus in novel coronavirus inactivated vaccine Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention provides a method for detecting the content of N protein of a novel coronavirus in a novel coronavirus inactivated vaccine. The detection method utilizes the quantitative peptide fragment (AYNVTQAFGR) of the invention; the mass-to-charge ratio of the quantitative ions of the quantitative peptide fragment to the parent ion is 563.7853, and the mass-to-charge ratio of the daughter ion is 679.3520 and/or 892.4640. And (3) utilizing the quantitative peptide segment and the quantitative ion pair, preparing a standard curve by an N 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 N 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 N protein of the new coronavirus in the intermediate product and the finished product of each stage of the inactivated vaccine of the new coronavirus.
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 N protein of novel coronavirus in the novel coronavirus inactivated vaccines, and particularly relates to a method for quantitatively detecting the content of N protein of novel coronavirus in the novel coronavirus inactivated vaccines by using liquid chromatography tandem mass spectrometry.
Background
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. Wherein, SARS-CoV-2 Nucleocapsid protein (N) is located in the virus, N protein is relatively conservative and accounts for the largest proportion in the virus structural protein, and in the process of assembling the virus body, the N protein is combined with virus RNA and leads to the formation of spiral Nucleocapsid. The N protein contains a large number of antigenic determinants that can generate protective antibodies against the virus. Studies have shown that the new coronavirus infection in the early stages of infection produces high levels of antibodies against the N protein. In addition, the nucleocapsid protein of SARS-CoV-2 is likely to induce specific T cell immune response, and has important effect on antagonizing virus infection.
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 N protein of the novel coronavirus in the inactivated vaccine needs to rely on an immunological analysis method, such as a double-antibody sandwich enzyme-linked immunosorbent assay.
The N protein in the novel coronavirus is detected by adopting an enzyme-linked immunosorbent assay technology, a recombinant N protein immune animal is utilized to prepare a specific monoclonal or polyclonal antibody, a double-antibody sandwich method is established, the specific N protein antibody is coated on an enzyme label plate, a sample to be detected is added for incubation, the N protein antibody marked with horseradish peroxidase is added for detection, and the N protein content of SARS-CoV-2 in a product to be detected can be quantitatively detected through a standard curve. In the technology for detecting N protein by an enzyme-linked method, the preparation of a specific N protein antibody is very critical, however, the preparation process of the antibody is time-consuming, and a high-sensitivity specific detection method cannot be established in a short time. Enzyme-linked immunosorbent assay (ELISA) is susceptible to environmental factors in the detection process due to its own limitations, so that the stability and repeatability are poor. The linear range of the external enzyme linked immunosorbent assay is narrow, and is generally 102For samples of unknown concentration, pre-experiments or sample dilutions are required to accurately determine that the sample falls within the optimal linear range.
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 N protein in the novel coronavirus inactivated vaccine, the invention aims to provide a quantitative peptide segment for quantitatively detecting the novel coronavirus N 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 N protein by liquid chromatography-tandem mass spectrometry; the third objective of the invention is to provide a method for detecting the content of the N protein of the novel coronavirus in the novel inactivated coronavirus vaccine, the quantitative peptide fragment and the quantitative ion pair of the N 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 N 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 N 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 N protein by liquid chromatography-tandem mass spectrometry, wherein the amino acid sequence of the quantitative peptide segment is as follows:
AYNVTQAFGR(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 (m/z) of the parent ion in the quantitative ion pair of the quantitative peptide fragment is 563.7853, and the mass-to-charge ratio (m/z) of the daughter ion is 679.3520 and/or 892.4640.
The quantitative peptide fragment and the quantitative ion pair thereof are applied to the quantitative detection of the novel coronavirus N 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 in a novel coronavirus inactivated vaccine sample on the detection, and can be used for detecting the content of the novel coronavirus N protein of intermediate products and finished products of each stage of 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 N protein by liquid chromatography-tandem mass spectrometry.
In another aspect, the present invention also provides a method for detecting the content of the N protein of the novel coronavirus in the inactivated vaccine of the novel coronavirus, which comprises the following steps:
incubating and performing enzymolysis treatment on an N protein standard substance of the recombinant novel coronavirus SARS-CoV-2; adopting high performance liquid chromatography to connect with triple quadrupole mass spectrometry detection equipment (HPLC-MRM-MS/MS), feeding an N 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 the peak area of a sub-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 N protein in the inactivated vaccine sample to be detected.
In the detection method, the N 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 N 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 N 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 of incubated N protein standard of 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 N 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 N 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) -28.0 of Q1 Pre of the quantitative ion pair having the parent ion mass-to-charge ratio of 563.7853 and the daughter ion mass-to-charge ratio of 679.3520; CE-19; q3 Pre was biased (v) -34.0.
Q1 Pre of ion pair 563.7853>892.4640 has a deviation (v) -30.0; CE-21; q3 Pre was biased (v) -26.0.
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 N 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 N 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 AYNVTQAFGR obtained by screening in example 1 of the present invention.
FIG. 2 is a secondary fragment mass spectrum of the quantitative peptide AYNVTQAFGR obtained from the screening in example 1 of the present invention.
FIG. 3 is a standard graph of a quantitative peptide AYNVTQAFGR of the N protein of the inactivated coronavirus vaccine of example 2.
FIG. 4 is the MRM map of the N protein quantitative peptide AYNVTQAFGR 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. The N protein standard of the recombined novel coronavirus SARS-CoV-2 and the inactivated vaccine sample of the novel coronavirus SARS-CoV-2 are processed:
taking an N protein standard product of the recombinant novel coronavirus SARS-CoV-2 or a novel coronavirus inactivated vaccine sample to be detected, adding a 0.1% RapidEST solution in an equal volume, incubating at 70 ℃ for 40min, then adding a 500mmol/L DTT solution to a final concentration of 25mmol/L, incubating at 65 ℃ for 40min, cooling to room temperature, adding a 1mol/L iodoacetamide solution to a final concentration of 50mmol/L, and reacting at room temperature in a dark place for 30 min. 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 the N protein sequence determined by mass spectrometry, 5 characteristic peptide sequences with the strongest detection signals and 19 pairs of ion pair mass spectrometry information (as shown in table 1 below) were obtained. Through validation analysis of Uniprot and NCBI databases, the 5 peptide fragment sequences are all N protein characteristic sequences.
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 N 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 N protein standard product and a virus culture solution, an intermediate product and a finished product of the 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 N 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 563.7853(m/z) >892.4640(m/z) and 563.7853(m/z) >679.3520(m/z) ion pairs of the AYNVTQAFGR peptide fragment have higher mass spectrum signal values for detecting the novel coronavirus N protein standard, the novel coronavirus inactivated vaccine virus culture solution, the intermediate product and the finished product, 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) AYNVTQAFGR 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 563.7853(m/z) >892.4640(m/z) and 563.7853(m/z) >679.3520(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 N protein of the novel coronavirus inactivated vaccine sample is finally determined to be AYNVTQAFGR, and the sequence of the quantitative ion pair is 563.7853(m/z) >679.3520(m/z) and/or 563.7853(m/z) >892.4640 (m/z). Fig. 1 is an ion flow diagram of AYNVTQAFGR peptide fragment, and fig. 2 is a secondary fragment mass spectrum of AYNVTQAFGR 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 N protein content of novel coronavirus in novel coronavirus inactivated vaccine
This example provides a method for detecting the content of N 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), 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) -28.0 for ion pair 563.7853(m/z) >679.3520 (m/z); CE-19; q3 Pre-bias (v) -34.0;
q1 Pre bias (v) -30.0 for ion pair 563.7853(m/z) >892.4640 (m/z); CE-21; q3 Pre was biased (v) -26.0.
The specific operation is as follows:
(1) adopting 50mmol/L NH to the N protein standard of the recombinant novel coronavirus SARS-CoV-24HCO3The solution was diluted to 16. 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 the incubated N protein standard4HCO3Adding trypsin solution 20 μ L, performing enzymolysis at 37 deg.C overnight, and adding formic acid 1 μ L after the enzymolysis reaction is completedSo as to inactivate the trypsin, namely finishing the enzymolysis reaction.
(2) Adopting high performance liquid chromatography to connect with triple quadrupole mass spectrometry detection equipment in series, injecting sample of N protein standard substance of the novel coronavirus SARS-CoV-2 with different volumes, 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 sub-ion of the quantitative peptide fragment ion pair 563.7853>892.4640 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＝156306X-26439，R2＝0.9983376，R＝0.9991684
(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. Then 50mmol/L NH is added 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 in figure 4 and a detection chart of 01 batches of samples) of 563.7853(m/z) >892.4640(m/z) of the quantitative peptide segment of the novel coronavirus SARS-CoV-2, and dividing the content by the sample injection volume to obtain the concentration of the novel coronavirus N 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 N protein of novel coronavirus in novel inactivated coronavirus vaccine
This example shows a process of methodological verification of the method for detecting the content of the N protein of the novel coronavirus in the inactivated vaccine by combining the quantitative peptide fragment and the quantitative ion of example 2 with a detection device of high performance liquid chromatography tandem triple quadrupole mass spectrometry.
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), 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) -28.0 for ion pair 563.7853(m/z) >679.3520 (m/z); CE-19; q3 Pre-bias (v) -34.0; q1 Pre bias (v) -30.0 for ion pair 563.7853(m/z) >892.4640 (m/z); CE-21; q3 Pre was biased (v) -26.0.
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 0.9983376, 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 the product when the signal to noise ratio of the daughter ion is greater than or equal to 3(S/N is greater than or equal to 3) is taken as the detection Limit (LODs). The detection limit and the quantification limit of the measured peptide fragment were 0.194. mu.g and 0.251. mu.g, respectively. The method is high in sensitivity and can meet the requirement of N protein detection.
(3) Precision: and (4) repeatedly carrying out sample injection and measuring the samples subjected to enzyme digestion treatment for 6 times by using 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 peptide fragment ion measured was 2.9%, indicating that the method was good in precision.
(4) And (3) accuracy, namely adding an N protein standard substance into the blank matrix of the new crown inactivated vaccine to be detected, detecting by the sample processing method, measuring the concentration of a standard sample by using a standard curve, and comparing an actual measurement value with a theoretical value to calculate the 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 95.7%, and the accuracy of the method was good.
Sequence listing
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<120> method for detecting content of N protein of novel coronavirus in novel coronavirus inactivated vaccine
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Claims (10)
1. A quantitative peptide segment for quantitative detection of novel coronavirus N protein by liquid chromatography-tandem mass spectrometry has the following amino acid sequence:
AYNVTQAFGR(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 563.7853, and the mass-to-charge ratio of the daughter ion is 679.3520 and/or 892.4640.
3. Use of the quantitative peptide fragment of claim 1 or 2 for the quantitative detection of the novel coronavirus N protein by liquid chromatography-tandem mass spectrometry.
4. A method for detecting the content of a novel coronavirus N protein in a novel coronavirus inactivated vaccine comprises the following steps:
incubating and performing enzymolysis treatment on an N protein standard substance of the recombinant novel coronavirus SARS-CoV-2; adopting high performance liquid chromatography to connect with triple quadrupole mass spectrometry detection equipment in series, feeding an N protein standard substance of the recombinant 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 a 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 N protein in the inactivated vaccine sample to be detected.
5. The detection method according to claim 4, wherein the incubation of the N 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 N 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 of incubated N protein standard of 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 N 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; and (3) carrying out gradient elution on the mobile phase B with the content changing from 0 to 45 percent in 0 to 20min, wherein the flow rate is 0.3 mL/min.
8. The method according to claim 4, wherein the mass spectrometric detection conditions of the N 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) -28.0 of Q1 Pre of a quantitative ion pair having a parent ion mass-to-charge ratio of 563.7853 and a child ion mass-to-charge ratio of 679.3520; CE-19; q3 Pre was biased (v) -34.0.
10. The detection method according to claim 4 or 8, wherein the deviation (v) -30.0 of Q1 Pre of a quantitative ion pair having a parent ion mass-to-charge ratio of 563.7853 and a child ion mass-to-charge ratio of 892.4640; CE-21; q3 Pre was biased (v) -26.0.
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| CN114324626A (en) * | 2021-11-08 | 2022-04-12 | 中国计量大学 | Characteristic peptide segment and method for detecting content of novel coronavirus spike protein |
| CN114702555A (en) * | 2022-03-09 | 2022-07-05 | 中国计量科学研究院 | Preparation and value determination method of novel coronavirus nucleocapsid protein (N protein) |
| WO2023123175A1 (en) * | 2021-12-30 | 2023-07-06 | 北京毅新博创生物科技有限公司 | Method for evaluating whether individual completes vaccination or individual immune changes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114324626A (en) * | 2021-11-08 | 2022-04-12 | 中国计量大学 | Characteristic peptide segment and method for detecting content of novel coronavirus spike protein |
| CN114324626B (en) * | 2021-11-08 | 2024-02-02 | 中国计量大学 | Characteristic peptide segment and method for detecting content of spike protein of novel coronavirus |
| WO2023123175A1 (en) * | 2021-12-30 | 2023-07-06 | 北京毅新博创生物科技有限公司 | Method for evaluating whether individual completes vaccination or individual immune changes |
| CN114702555A (en) * | 2022-03-09 | 2022-07-05 | 中国计量科学研究院 | Preparation and value determination method of novel coronavirus nucleocapsid protein (N protein) |
| CN114702555B (en) * | 2022-03-09 | 2023-01-03 | 中国计量科学研究院 | Preparation and value determination method of novel coronavirus nucleocapsid protein N protein |
| WO2023169246A1 (en) * | 2022-03-09 | 2023-09-14 | 中国计量科学研究院 | Method for preparing and certifying sars-cov-2 nucleocapsid protein (n protein) |
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