Specific polypeptide of HA protein in H5N1 avian influenza vaccine and quantitative detection method

Abstract

The invention relates to the field of virology. In particular, the invention relates to a polypeptide for quantitatively detecting the HA protein of H5N1, a kit containing the polypeptide, and a method for quantitatively detecting the HA protein of H5N1 by using the polypeptide. The method is a quantitative method with high throughput, high selectivity and high sensitivity, and can be used for absolute quantification of HA protein in H5N1 vaccine virus liquid.

Description

Technical Field

[0001] This invention relates to the field of virology. In particular, this invention relates to a polypeptide for the quantitative detection of the HA protein of H5N1, a kit containing said polypeptide, and a method for the quantitative detection of the HA protein of H5N1 using said polypeptide. Background Technology

[0002] H5N1 highly pathogenic avian influenza is an infectious disease of birds caused by avian influenza viruses. It typically does not infect humans, but when the virus's antigenicity changes, it can cause severe illness and death, leading to a major public health problem. From 2020 to 2021, the H5N1 clade 2.3.4.4b virus spread widely among wild birds globally, causing outbreaks in poultry and other animals. In 2024, the United States reported the world's first case of "cattle-to-human" transmission, with the patient exhibiting conjunctivitis or mild respiratory symptoms; however, studies have shown that the virus is fatal to ferrets and mice.

[0003] Vaccination is a key strategy for preventing and controlling influenza pandemics. China's first H5N1 influenza vaccine, "Pan'erlaifu," was developed by Beijing Sinovac Biotech Co., Ltd. in 2004. A 10 μg dose of the vaccine achieved a seroconversion rate of 78.3%, demonstrating good immunogenicity and safety, and gained international recognition. The main immunogenic protein in pandemic vaccines is hemagglutinin (HA). The single radial immune diffusion assay (SRID) is a recognized method for detecting HA content. It uses an agarose gel plate containing antiserum of a specific strain; after punching holes, an antigen standard containing the corresponding strain is added. The diameter of the resulting precipitate ring is proportional to the concentration of the standard; the HA content can be calculated from the sample diameter. The WHO General Protocol for Calibration of Working Reagents for Seasonal and Pandemic Influenza Antigens in Basic Regulatory Laboratories specifies the calibration methods for antigen standards and antiserum reagents. For antigen standards, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Lowry method are used to quantify the HA content of whole virus solutions. Then, sheep serum immunized with purified HA is used to perform SRID experiments to determine the HA content of the lyophilized antigen standards. However, the Lowry method is susceptible to interference from ions, buffers, and reducing agents, especially the sucrose used in virus purification, which significantly affects the detection results. Furthermore, SDS-PAGE has limited resolution and sensitivity; low-abundance protein bands are easily masked by the main band, making high-precision purity analysis difficult. In the whole virus solution produced by MDCK cell matrix, the host protein bands overlap with the HA bands, resulting in a higher HA purity value detected by SDS-PAGE. Therefore, traditional methods cannot be used to calibrate the HA antigen standards for this matrix-based influenza vaccine, hindering the development and market launch of the MDCK cell matrix influenza vaccine.

[0004] In recent years, numerous new methods for detecting hemagglutinin content have emerged, including HPLC, LAA, SPRi, LC-MS, and VaxArray analytical platforms. Among these, Barr et al. first proposed using proteolytic enzymes combined with isotopic dilution mass spectrometry (IDMS) to quantify proteins in 1996. This method is currently widely used in food safety, clinical drug analysis, and vaccine quality evaluation. In quantitative studies of standards for seasonal and pandemic influenza vaccines, the combination of IDMS and MRM modes allows 1 mol of specific peptides to represent 1 mol of target protein. By screening for specific peptides after target protease digestion and adding isotopically labeled internal standard peptides to the sample, the peak area ratio of the specific peptides and internal standard peptides in the sample can be used to correct for losses during pretreatment and analysis. This allows for accurate quantification of hemagglutinin (HA) in avian influenza vaccine virus solution without the need for antiserum reagents. Traditional methods for preparing immune serum require 2–3 months, while peptide synthesis can be completed in only 2–3 weeks, significantly shortening the standard calibration time and accelerating vaccine market launch.

[0005] Currently, the globally prevalent strains are branch 2.3.4.4b (such as A / Texas / 37 / 2024). The WHO's newly released list of H5N1 candidate vaccine strains includes the locally prevalent Chinese strain A / Jiangsu / NJ210 / 2023 (hereinafter referred to as the Jiangsu strain). Therefore, based on the needs of pandemic prevention and control, it is necessary to screen for specific peptides of H5N1 (including the Jiangsu strain) and establish a high-throughput, high-selectivity, and high-sensitivity IDMS quantitative method. Summary of the Invention

[0006] To address the aforementioned problems, the inventors of this application, through extensive experimentation, screened out novel specific peptides suitable for determining the HA protein content of broad-spectrum H5N1. Furthermore, based on this specific peptide and isotope dilution mass spectrometry, a method for determining the HA protein content in H5N1 vaccines was established. Moreover, this invention is the first to propose introducing an extended internal standard peptide (i.e., the third peptide described below) into the method to simulate the enzymatic cleavage effect of real HA protein, reducing the influence of the HA protein mixture (e.g., matrix effect, adjuvant effect), and further improving detection performance (e.g., precision, accuracy). Thus, the inventors completed this application.

[0007] Therefore, in one aspect, the present invention provides a polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0008] In some embodiments, the polypeptide is unlabeled.

[0009] In some embodiments, the polypeptide has the amino acid sequence shown in SEQ ID NO: 1, which is used to determine the amount of H5N1 HA protein in a sample. In some embodiments, the polypeptide has the amino acid sequence shown in SEQ ID NO: 2 or 3, which is used to determine the amount of H5N1 HA protein in a sample. In some embodiments, the sample is an H5N1 vaccine or vaccine intermediate (e.g., vaccine viral solution) containing H5N1 HA protein. In some embodiments, the sample is a cell matrix (e.g., MDCK cell matrix) or chicken embryo matrix vaccine viral solution containing H5N1 HA protein.

[0010] In this invention, the polypeptides of this invention are not limited to any particular method of synthesizing polypeptides and can be generated by conventional techniques known to those skilled in the art, such as DNA recombination or chemical synthesis. In some embodiments, the polypeptides of this invention are generated by chemical synthesis. Methods for total chemical synthesis of proteins or polypeptides are well known in the art (see, for example, Raibaut L, et al., Top Curr Chem. 2015; 363:103-54; Thapa P, et al. Molecules. 2014; 19(9):14461-83; Dawson PE, et al., Science, 1994; 266(5186):776-9; these are incorporated herein by reference), and include, but are not limited to: solid phase peptide synthesis (SPPS) or liquid phase fractional synthesis (e.g., native chemical linkage (NCL), azide method, transfer active ester condensation (TAEC)). Alternatively, the polypeptides of the present invention can be obtained by DNA recombination technology, for example, by using a cell-free expression system from polynucleotides encoding these proteins or polypeptides (cell-free expression systems include, for example, reticulocyte lysate-based expression systems, wheat germ extract-based expression systems, and Escherichia coli extract-based expression systems); or by using an in vivo expression system (e.g., Escherichia coli prokaryotic expression system, yeast eukaryotic expression system) from polynucleotides encoding these proteins or polypeptides.

[0011] In some embodiments, the polypeptide is either unlabeled or labeled with a detectable tag.

[0012] In some embodiments, the polypeptide is labeled with an isotope.

[0013] In some embodiments, one or more (e.g., 1, 2, or 3) amino acid residues in the polypeptide are labeled with stable isotopes.

[0014] In some embodiments, the stable isotope label is selected from... 2 H, 13 C 15 N、 17 O、 18 O, or any combination thereof.

[0015] In some embodiments, the stable isotope is labeled as 13 C and / or 15 N.

[0016] In some embodiments, the polypeptide has the following sequence: A[I]DGVTNK (SEQ ID NO: 1), where [I] indicates that one or more atoms in the isoleucine are replaced by their corresponding stable isotopes.

[0017] In some embodiments, all carbon atoms in the isoleucine are... 13 C is replaced, and all nitrogen atoms are replaced. 15 N is replaced.

[0018] Preferably, the sample is an H5N1 vaccine virus solution containing the H5N1 HA protein.

[0019] In another aspect, the present invention provides an isolated nucleic acid that encodes a polypeptide as described above.

[0020] In another aspect, the present invention provides a vector comprising the isolated nucleic acid as described above. Vectors suitable for inserting a target polynucleotide are well known in the art, including but not limited to cloning vectors and expression vectors. In some embodiments, the vector is, for example, a plasmid, a granule, a bacteriophage, etc.

[0021] In another aspect, the present invention also relates to host cells comprising the isolated nucleic acids or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, e.g., primate cells, human cells, etc.). The host cells of the present invention can also be cell lines, such as 293T cells.

[0022] In another aspect, the present invention provides a kit comprising:

[0023] A first polypeptide having the amino acid sequence shown in SEQ ID NO: 1, and the first polypeptide being unlabeled; and / or

[0024] The second polypeptide has the same amino acid sequence as the first polypeptide and is labeled with an isotope.

[0025] In some preferred embodiments, the kit is used to determine the amount of H5N1 HA protein in a sample. In such embodiments, the first polypeptide serves as a specific external standard peptide, and the second polypeptide serves as an isotope-labeled internal standard peptide, which can be used to establish a standard curve and determine the amount of H5N1 HA protein in the sample.

[0026] In some embodiments, one or more (e.g., one, two, or three) amino acid residues in the second polypeptide are labeled with a stable isotope. In some embodiments, the stable isotope label is selected from... 2 H, 13 C 15 N、 17 O、 18 O, or any combination thereof. In some embodiments, the stable isotope is labeled as... 13 C and / or 15 N.

[0027] In some embodiments, the second polypeptide has the following sequence: A[I]DGVTNK (SEQ ID NO: 1), where [I] indicates that one or more atoms in the isoleucine are replaced by their corresponding stable isotopes. In some embodiments, all carbon atoms in the isoleucine are replaced by... 13 C is replaced, and all nitrogen atoms are replaced. 15 N is replaced.

[0028] In some embodiments, the kit described above further comprises:

[0029] The third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5, and the third polypeptide is labeled with an isotope.

[0030] In some embodiments, the third polypeptide further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids at both ends of the amino acid sequence shown in SEQ ID NO: 5, and is a truncated peptide of the H5N1 HA protein.

[0031] It is understood that the third polypeptide is an elongated second polypeptide. Although it contains additional amino acids at both ends of the amino acid sequence shown in SEQ ID NO: 1, these additional amino acids are not arbitrary. Since the third polypeptide is a truncated peptide of the H5N1 HA protein, the additional amino acids are those located at both ends of the amino acid sequence shown in SEQ ID NO: 1 in the HA protein.

[0032] In some embodiments, the third polypeptide has the amino acid sequence shown in SEQ ID NO: 5.

[0033] In some embodiments, one or more (e.g., one, two, or three) amino acid residues in the third polypeptide are labeled with a stable isotope. In some embodiments, the stable isotope label is selected from... 2 H, 13 C 15 N、 17 O、 18 O, or any combination thereof. In some embodiments, the stable isotope is labeled as... 13 C and / or 15 N.

[0034] In some embodiments, the third polypeptide has the following sequence: QKA[I]DGVTNKVN (SEQ ID NO: 5), where [I] indicates that one or more atoms in the isoleucine are replaced by their corresponding stable isotopes. In some embodiments, all carbon atoms in the isoleucine are replaced by... 13 C is replaced, and all nitrogen atoms are replaced. 15 N is replaced.

[0035] In some embodiments, the kit further includes reagents for mass spectrometry detection of the first polypeptide, the second polypeptide, and / or the third polypeptide.

[0036] In some embodiments, the kit further comprises reagents for pretreatment of the sample, the first polypeptide, the second polypeptide, and / or the third polypeptide.

[0037] In some embodiments, the kit also contains a denaturing agent (e.g., urea, guanidine hydrochloride, or an ionic surfactant).

[0038] In some embodiments, the kit also contains a reducing agent (e.g., dithiothreitol, mercaptoethanol, or TECP).

[0039] In some embodiments, the kit also contains an alkylating agent (e.g., iodoacetamide).

[0040] In some embodiments, the kit further comprises a protease (e.g., trypsin, LysC protease, GluC protease, AspN protease, or chymotrypsin).

[0041] In some embodiments, the kit also contains water (e.g., pure water, ultrapure water, deionized water, or distilled water).

[0042] In some embodiments, the denaturant is Rapidus SF.

[0043] In another aspect, the present invention also relates to a method for determining the amount of H5N1 HA in a sample, comprising the step of determining the amount of a first polypeptide in an enzymatic digestion product of the sample using mass spectrometry, wherein the first polypeptide has the amino acid sequence shown in SEQ ID NO: 1 and is unlabeled.

[0044] In this invention, the method of determining the amount of the first polypeptide in the enzyme digestion product of the sample using mass spectrometry is well known in the art, and examples include, but are not limited to, those described in detail in "Guoan Zhang et al., Methods MolBiol. 2010; 673:211–222." In some preferred embodiments, isotope dilution mass spectrometry (IDMS; see, for example, WO 03 / 016861) is used to determine the amount of the first polypeptide in the enzyme digestion product of the sample.

[0045] In some embodiments, the amount of the first polypeptide in the enzyme digestion product of the sample is determined by isotope dilution mass spectrometry (IDMS).

[0046] In some embodiments, the enzyme digestion product is a trypsin digestion product.

[0047] In some embodiments, the sample is treated with a denaturant before being enzyme digested.

[0048] In some embodiments, a step of purifying the enzyme digestion product is included before determining the amount of the first polypeptide in the enzyme digestion product of the sample using mass spectrometry. In some embodiments, the purification is chromatographic, such as liquid chromatography (e.g., high-performance liquid chromatography or ultra-high-performance liquid chromatography).

[0049] In some preferred embodiments, the purification is selected from filtration, chromatography (e.g., gas chromatography or liquid chromatography, such as high-performance liquid chromatography or ultra-high-performance liquid chromatography), electrophoresis (e.g., gel electrophoresis or capillary electrophoresis), or any combination thereof. In some preferred embodiments, the purification is chromatography, such as liquid chromatography. In some preferred embodiments, the purification is high-performance liquid chromatography or ultra-high-performance liquid chromatography.

[0050] In some preferred embodiments, the mass spectrometer is selected from quadrupole mass spectrometry, ion trap mass spectrometry, time-of-flight mass spectrometry, or tandem mass spectrometry. In some preferred embodiments, the mass spectrometer is a tandem mass spectrometer, such as triple quadrupole mass spectrometry (QqQ) or quadrupole time-of-flight tandem mass spectrometry (QTOF). In one specific embodiment, the mass spectrometer is a triple quadrupole mass spectrometer.

[0051] In some preferred embodiments, the detection mode of the mass spectrometer is single ion monitoring (SIM), selected reaction monitoring (SRM), multiple reaction monitoring (MRM), or multiple selected reaction monitoring (mSRM). In one specific embodiment, the detection mode of the mass spectrometer is MRM.

[0052] In some embodiments, the sample is an H5N1 cell matrix containing the H5N1 HA protein (e.g., MDCK cell matrix) or a chicken embryo matrix vaccine virus solution.

[0053] In some implementations, the method includes:

[0054] (i) The sample is digested using a protease to obtain the enzyme digestion product;

[0055] (ii) Perform mass spectrometry analysis on the enzyme digestion product obtained in step (i) and obtain the signal intensity of the first polypeptide;

[0056] (iii) Compare the signal intensity obtained in step (ii) with a standard curve and obtain the molar content of the first polypeptide, which is the amount of H5N1 HA protein in the sample; wherein the standard curve is a linear relationship between the known amount of the first polypeptide and the signal intensity.

[0057] In some preferred embodiments, the protease is trypsin.

[0058] In some preferred embodiments, the signal strength is a peak height or a peak area. In some preferred embodiments, the signal strength is a peak area.

[0059] In some preferred embodiments, prior to step (ii), a step of purifying the enzyme digestion product is included. In some preferred embodiments, the purification is by chromatography, such as liquid chromatography. In some preferred embodiments, the purification is by high-performance liquid chromatography or ultra-high-performance liquid chromatography.

[0060] In some preferred embodiments, in step (ii), the mass spectrometer is a tandem mass spectrometer, such as a triple quadrupole mass spectrometer (QqQ) or a quadrupole time-of-flight tandem mass spectrometer (QTOF). In one specific embodiment, the mass spectrometer is a triple quadrupole mass spectrometer. In another specific embodiment, the mass spectrometry mode in the mass spectrometer is MRM.

[0061] In some preferred embodiments, in step (ii), the mass spectrometry analysis includes the following steps:

[0062] The enzyme digestion product obtained in step (i) is ionized to obtain the parent ion and daughter ion of the first polypeptide; the signal intensity of the obtained daughter ion is measured, and the signal intensity is the signal intensity of the first polypeptide.

[0063] In some preferred embodiments, the ionization method is ESI.

[0064] In some implementations, the method includes:

[0065] (1) The sample was digested using a protease to obtain the enzyme digestion product;

[0066] (2) Add a known amount of the second polypeptide to the enzyme digestion product obtained in step (1) and obtain a mixture;

[0067] (3) Perform mass spectrometry analysis on the mixture obtained in step (2) and obtain the signal intensities of the first polypeptide and the second polypeptide;

[0068] (4) Calculate the ratio of the signal intensity of the first peptide to that of the second peptide, and calculate the ratio of the concentration of the first peptide to that of the second peptide;

[0069] (5) Compare the ratio with the standard curve and obtain the content of the first polypeptide, which is the amount of H5N1 HA protein in the sample; wherein the standard curve is a linear relationship between the concentration ratio of the first polypeptide and the signal intensity ratio.

[0070] In some embodiments, the protease is trypsin.

[0071] In some embodiments, in step (b), the ratio of the mixture to the protease is 2:1 to 1:1; for example, 2:1, 1.75:1, 1.5:1, 1.25:1, or 1:1. In some embodiments, in step (b), the digestion time is 1-4 hours; for example, 1 hour, 2 hours, 3 hours, or 4 hours.

[0072] In some embodiments, a series of first peptide samples with known concentrations are prepared, and a second peptide at a fixed known concentration is added to each first peptide sample. Each mixed sample is then subjected to mass spectrometry analysis to measure the signal intensities (e.g., peak areas) of the first and second peptides, and the signal intensity ratio R and concentration ratio C of the first and second peptides are calculated. A standard curve is plotted with R on the ordinate and C on the abscissa.

[0073] In some preferred embodiments, the signal strength is a peak height or a peak area. In some preferred embodiments, the signal strength is a peak area.

[0074] In some preferred embodiments, a step of purifying the enzyme digestion product is included prior to step (3). In some preferred embodiments, the purification is by chromatography, such as liquid chromatography. In some preferred embodiments, the purification is by high-performance liquid chromatography or ultra-high-performance liquid chromatography.

[0075] In some preferred embodiments, in step (3), the mass spectrometry analysis is triple quadrupole mass spectrometry. In some preferred embodiments, in step (3), the detection mode of the mass spectrometry analysis is MRM. In some preferred embodiments, the ionization method is ESI.

[0076] In some embodiments, the method includes:

[0077] (a) Providing a mixture comprising a known amount of a first polypeptide and a second polypeptide, performing mass spectrometry analysis on the mixture, and obtaining the signal intensities of the first polypeptide and the second polypeptide; wherein the second polypeptide has the same amino acid sequence as the first polypeptide and is labeled with an isotope;

[0078] Then, based on the signal strength, a standard curve is plotted;

[0079] (b) Providing a mixture comprising a known amount of a third polypeptide and a sample comprising H5N1 HA protein; wherein the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 and is labeled with an isotope;

[0080] The mixture was digested using a protease to obtain an enzyme digestion product containing unknown amounts of a first polypeptide and a second polypeptide; the enzyme digestion product was then analyzed by mass spectrometry to obtain the signal intensity of the unknown amounts of the first polypeptide and the second polypeptide.

[0081] (c) Compare the signal intensities of the first and second peptides obtained in step (b) with the standard curve, and analyze the content of the unknown amount of the first peptide, which is the amount of H5N1 HA protein in the sample.

[0082] Steps (a) and (b) are not in any particular order.

[0083] In the above embodiment, in step (b), since the third polypeptide will generate a second polypeptide after enzymatic digestion, the digestion product will only contain the first polypeptide and the second polypeptide. Furthermore, by comparing the content of the third polypeptide provided in step (b) with the content of the second polypeptide generated after its enzymatic digestion, the amount of third polypeptide lost during the digestion process can be determined. This amount of loss can be used to calibrate the amount of first polypeptide lost from the HA protein in the sample during the digestion process.

[0084] It is understandable that the content of H5N1HA protein in a sample can be detected using only the first and second peptides without the use of the third peptide. However, the detection performance (e.g., precision, accuracy) can be improved by adding the third peptide. Therefore, the above detection method is not only suitable for detecting finished vaccines, but also particularly suitable for detecting vaccine intermediates in the production process (e.g., cell matrix containing H5N1HA protein (e.g., MDCK cell matrix) or samples of chicken embryo matrix vaccine viral fluid).

[0085] In some embodiments, in step (a), a linear regression analysis is performed using the peak area ratio of the first peptide and the second peptide and the corresponding concentration ratio of the first peptide and the second peptide to obtain a standard curve of the concentration of the first peptide relative to the peak area ratio (e.g., the first peptide and / or the second peptide). Then, the signal intensities of the first peptide and the second peptide obtained in step (b) are compared with the standard curve. Based on the amount of the third peptide before enzymatic digestion and the amount of the second peptide produced after digestion, the content of the first peptide in the sample is calibrated, and the content of the unknown amount of the first peptide is analyzed, which is the amount of H5N1 HA protein in the sample.

[0086] In some embodiments, the protease is trypsin.

[0087] In some embodiments, in step (b), the ratio of the mixture to the protease is 2:1 to 1:1; for example, 2:1, 1.75:1, 1.5:1, 1.25:1, or 1:1. In some embodiments, in step (b), the digestion time is 1-4 hours; for example, 1 hour, 2 hours, 3 hours, or 4 hours.

[0088] In some preferred embodiments, the signal strength is a peak height or a peak area. In some preferred embodiments, the signal strength is a peak area.

[0089] In some preferred embodiments, step (b) further includes a step of purifying the enzyme digestion product. In some preferred embodiments, the purification is by chromatography, such as liquid chromatography. In some preferred embodiments, the purification is by high-performance liquid chromatography or ultra-high-performance liquid chromatography.

[0090] Terminology Definition

[0091] As used herein, the term "H5N1 HA protein" refers to the hemagglutinin (HA) protein in the H5N1 influenza A virus (also known as "avian influenza virus"), which is one of the major antigens of the avian influenza virus. The amino acid sequence of the HA protein is well known in the art and can be found in various public databases (e.g., Gisaid). In some embodiments, the sequence of the HA protein is as shown in SEQ ID NO: 8 or 9.

[0092] As used herein, the term "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, β-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, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers.

[0093] As used herein, the term "stable isotope labeling" refers to a labeling of an isotope that does not undergo radioactive decay or whose radioactive decay cannot be measured by current detection techniques. Such stable isotopes are well known in the art, and examples include, but are not limited to, those listed below. 2 H, 13 C 15 N、 17 O、 18 O、 33 S, 34 S. In this invention, the expression "a polypeptide with a stable isotope label" means that the amino acid sequence of the polypeptide contains at least one amino acid in which at least one atom (e.g., C, H, O, N, or S) is replaced by its stable isotope. Methods for obtaining polypeptides with stable isotope labels are well known in the art, such as metabolic labeling methods and chemical labeling methods (see, for example, Zhang Jing et al., Organic Chemistry, Vol. 31, No. 12, 2011, pp. 2043-2051).

[0094] As used herein, the term "isotope dilution mass spectrometry (IDMS)" has the meaning commonly understood by those skilled in the art, and its procedures can be found in, for example, Barcelona-Barrachina, E. et al. Journal of chromatography. A 2006, 1125, 195-203; Mezcua, M. et al. Journal of chromatography. A 2006, 1109, 222-227; Wang, X. et al. Analytica chimica acta 2007, 594, 265-273; Zhang, Y. et al. Journal of chromatography. A 2007, 1142, 194-198; Yu, K. et al. Rapid communications in mass spectrometry: RCM 2007, 21 ,893-902; Williams, TL. et al. Vaccine 2008, 26, 2510-2520; Williams, T.L. et al. Vaccine 2012, 30, 2475-2482; Luna, L.G. et al. Analytical Chemistry 2008, 80, 2688-2693; and International Patent Application WO 03 / 016861; all of which are incorporated herein by reference.

[0095] As used herein, the term "enzyme digestion product" refers to the hydrolysate obtained by digesting a protein component in a sample with a protease, which contains amino acid fragments of the protein in the sample. In this invention, the expression "trypsin digestion product" means the hydrolysate obtained by digesting a protein component in a sample with trypsin.

[0096] As used herein, the term "purification" refers to the process of enriching an amount of one or more analytes relative to one or more other components in a sample. Therefore, those skilled in the art will understand that purification as described in this invention does not mean the complete removal of all components from a sample except for the analyte (e.g., the characteristic peptide or internal standard peptide as described herein), and purification as described in this invention does not necessarily require the separation of the analyte from all other components. In some preferred embodiments, the purification may be used to remove one or more interfering substances that may interfere with the detection of the analyte ions by mass spectrometry. Methods that can be used for purification are well known in the art, and examples include, but are not limited to, filtration, chromatography (e.g., gas chromatography or liquid chromatography, such as high performance liquid chromatography or ultra-high performance liquid chromatography), or electrophoresis (e.g., gel electrophoresis or capillary electrophoresis).

[0097] As used herein, the term "denaturant" refers to a substance that can inhibit the formation of secondary and tertiary structures of proteins and prevent interactions between proteins. Such substances are well known in the art, and examples include, but are not limited to, urea, thiourea, guanidine hydrochloride, or ionic surfactants, wherein the ionic surfactants are described in detail in U.S. patent applications US7229539 and US8580533, the entire contents of which are incorporated herein by reference.

[0098] As used herein, the term "reducing agent" refers to a substance that can break the disulfide bonds of a protein, thereby reducing the protein and preventing its oxidation. Such substances are well known in the art, and examples include, but are not limited to, dithiothreitol (DTT), β-mercaptoethanol, or TCEP.

[0099] As used herein, the term "alkylating agent" has the meaning commonly understood by those skilled in the art. In this invention, the alkylating agent preferably refers to a reagent capable of reacting with the free sulfhydryl group (SH-) in an amino acid (e.g., cysteine) to prevent its oxidation and subsequent formation of a disulfide bond; such reagents are well known in the art, for example, iodoacetic acid, iodoacetamide, etc.

[0100] Beneficial effects of the invention

[0101] This invention provides a novel characteristic peptide for determining the HA protein content of H5N1, and a method for determining the HA protein content in H5N1 seedlings based on this characteristic peptide and isotope dilution mass spectrometry. Furthermore, this invention is the first to propose introducing an isotope-labeled extended internal standard peptide into the method to simulate the enzymatic cleavage effect of real HA protein, reduce the influence of matrix effects, and improve detection performance (e.g., precision, accuracy).

[0102] First, compared to traditional methods (e.g., SRID), the method of this invention uses a characteristic peptide without the need for specific antibodies, making the detection method faster and more convenient. In particular, since the Jiangsu strain is a newly emerging circulating strain, the standard antigen and antiserum reagents corresponding to the vaccine strain are not yet commercially available. Therefore, for newly emerging strains, traditional methods require spending considerable time and money to manufacture specific antibodies before performing SRID detection.

[0103] Secondly, the detection performance of the characteristic peptides and detection methods of the present invention is comprehensively improved. For example, the intra-day and inter-day precision CV values ​​are all below 15%, indicating good precision. For example, compared with the results of SRID (the internationally recognized gold standard for detection), the concordance rate is as high as 85%-101%, indicating good accuracy. For example, compared with SRID (the internationally recognized gold standard for detection), the sensitivity is improved by 6.7 times.

[0104] Finally, the characteristic peptides and detection methods of the present invention can simultaneously achieve accurate quantification of key proteins such as HA and NA of H5N1, and there is no cross-interference between the ion channels, exhibiting extremely strong specificity.

[0105] Therefore, the HA protein quantitative detection method based on characteristic peptides of H5N1 of the present invention has significant and advantageous technical effects and is easy to popularize.

[0106] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0107] Figure 1 The amino acid sequence is that of the H5N1 strain from Jiangsu.

[0108] Figure 2 This is a TIC chromatogram of a specific peptide. Figure 2 The peptide information is as follows: H5-1: AIDGVTNK; H5-4: TLDFHDSNVK; H5-7: LVLATGLR; H5-2: AIDGVTNK; H5-5: TLDFHDSNVK; H5-8: LVLATGLR.

[0109] Figure 3 The figure shows the results of optimizing the enzyme digestion ratio and digestion time; among them, Figure 3 The chicken embryo matrix whole virus solution in A and C are samples from different batches.

[0110] Sequence information

[0111] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0112] Table 1: Sequence Description Detailed Implementation

[0113] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0114] Unless otherwise specified, the experiments and methods described in the examples are generally performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); and the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL*. APPROACH (edited by MJ MacPherson, BD Hames and GR Taylor (1995)), Harlow and Lane (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by R.R. Freshney (1987)).

[0115] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0116] Example 1. Experimental Method

[0117] The applicant of this application first analyzed and screened specific peptides in the HA protein of H5N1, then labeled them with isotopes to serve as internal standard peptides, and combined this with IDMS to detect the HA content in the H5N1 vaccine. Furthermore, the applicant developed an IDMS method based on this specific peptide to detect HA content, and compared the detection concordance rate of this method with internationally recognized HA content detection methods SRID and SDS-PAGE+Lowry.

[0118] vaccine samples

[0119] Whole virus solution and lysis stock solution of MDCK cell matrix from strain A / Vietnam / 1194 / 2004 (hereinafter referred to as the Vietnamese strain), whole virus solution of MDCK cell matrix from the Jiangsu strain, and negative samples of cell culture medium were all provided by Wuhan Institute of Biological Products Co., Ltd. MDCK cell source samples were inoculated with influenza vaccine strains, treated with nucleases, and then inactivated and purified to obtain whole virus solution, which was subsequently lysed to obtain lysis stock solution. Whole virus solution of chicken embryo matrix and negative samples of chicken embryo allantoic fluid from the Jiangsu strain were provided by Beijing Sinovac Biotech Co., Ltd. The whole virus solution was obtained by inactivation and purification of chicken embryo harvest fluid.

[0120] SRID Experiment

[0121] Reagents and Instruments

[0122] Standard antigens and antiserum reagents were obtained from NIBSC: Antigen 09 / 184: A / Vietnam / 1194 / 2004 (NIBRG-14), Antiserum 07 / 148: A / Vietnam / 1194 / 04. Agarose was purchased from Sigma-Aldrich, Zwittergent 3-14 detergent from Merck, and Coomassie Brilliant Blue from Adamas-beta. The influenza hemagglutinin content scanning analysis system was purchased from Microvision.

[0123] Experimental methods

[0124] According to the instructions for use of the antigen standard, it was diluted with pure water to a concentration of 10–40 μg / ml. The sample was then diluted twice with pure water and added to agarose gel plates containing an appropriate concentration of antiserum reagent. After being placed at room temperature for 18 to 24 hours, the sample was soaked, dried, stained, and decolorized. The diameter of the precipitation ring was measured using a hemagglutinin content scanning analysis system. The HA content of the sample was calculated based on the linear regression equation.

[0125] Lowry Experiment

[0126] Reagents and Instruments

[0127] Serum albumin standards (bovine) were obtained from NIFDC; potassium tartrate and copper sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd.; and Folin-Ciocalteu was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The enzyme-linked immunosorbent assay (ELISA) instrument was purchased from Molecular Devices.

[0128] Experimental methods

[0129] Dissolve the standard in water to prepare a standard solution of 0–200 μg / ml. Dilute the sample 10-fold with pure water. Add 1 ml of alkaline copper solution, shake well, and let stand at room temperature for 10 minutes. Add 4 ml of Folin-Ciocalteu solution to each sample, mix immediately, and let stand at room temperature for 30 minutes. Measure the absorbance at 650 nm. Calculate the regression equation using the standard solution concentration and its corresponding absorbance. Calculate the protein concentration of the sample based on the absorbance.

[0130] SDS-PAGE Experiment

[0131] Reagents and Instruments

[0132] Protein Marker was purchased from Thermo Fisher Scientific, PNGase F kit from New England Biolabs, sample buffer from Bio-Rad, and gel imaging analysis system from Cytiva.

[0133] Experimental methods

[0134] According to the PNGase F kit instructions, the samples were denatured, reduced, and deglycosylated before protein gel electrophoresis. After fixation, staining, and destaining, the gels were photographed using a gel imaging analysis system and analyzed using grayscale analysis software. The glycosidase F band was removed, and the HA purity was calculated after the bands were normalized by area.

[0135] IDMS Experiment

[0136] Reagents, consumables and instruments

[0137] The reducing agent RapiGest SF was purchased from Waters, mass spectrometry-grade trypsin was purchased from Promega, mass spectrometry-grade formic acid, acetonitrile, and purified water were purchased from Thermo Fisher Scientific, the chromatographic column ACQUITY BEH C18 (2.1*100 mm, 1.7 μm) was purchased from Waters, the high-resolution liquid chromatography-mass spectrometry system Orbitrap was purchased from Thermo Fisher Scientific, and the triple quadrupole LCMS-8050 system was purchased from Shimadzu Corporation. All pipette tips, EP tubes, and sample vials used in the experiments were made of protein-low adsorption material.

[0138] Peptide screening

[0139] The H5N1 amino acid sequences of 34 WHO-recommended vaccine candidate strains and circulating strains, including the Jiangsu strain (SEQ ID NO: 8), the Vietnam strain (SEQ ID NO: 9), and the Texas strain, were downloaded from the Gisaid website. Protein Prospector software was used to theoretically digest the peptides at the lysine (K) and arginine (R) positions to screen for theoretical peptides and predict their parent and daughter ions. After denaturation, reduction, alkylation, and enzyme digestion, vaccine samples were analyzed using liquid chromatography-high-resolution mass spectrometry (LC-MS / MS). Peaks software was used to analyze the data and compare it with the theoretical enzyme digestion peptide information to screen for specific peptides.

[0140] Peptide synthesis and formulation

[0141] Specific external standard peptides, isotope-labeled internal standard peptides, and isotope-labeled extended internal standard peptides (with two amino acids added to each side of the internal standard peptide according to the theoretical sequence) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (sequence information is shown in Tables 1 and 2). After desalting, the peptides were in the form of white lyophilized powder, with HPLC purity greater than 95%. A stock solution was prepared by dissolving 1 mg of peptide in 1 ml of pure water. Based on the peptide molecular weight and purity specified in the instructions, the stock solution was diluted with pure water to prepare a 10 nM / ml working solution. After aliquoting, the solution was stored at -80℃ for the preparation of standard curves.

[0142] Table 2. Specific peptide sequences and MRM mass spectrometry acquisition methods

[0143] Note: The underlined amino acids are... 13 C and 15 N isotope-labeled amino acids.

[0144] Take 8 μl of 10 nM / ml internal standard working solution and add 392 μl of pure water to obtain a 200 pM / ml internal standard solution. Take another 120 μl of the 200 pM / ml internal standard solution and add 480 μl of pure water to dilute to a 40 pM / ml internal standard solution. Separately, serially dilute the 10 nM / ml external standard working solution with 0.1% formic acid to concentrations of 1000, 400, 200, 100, 50, 10, 5, and 1 pM / ml. Take 60 μl of each of these solutions, add 60 μl of the internal standard solution (40 pM / ml), vortex to mix, and transfer to a vial. The final standard curve concentrations of the external standard peptide are 0.5, 2.5, 5, 25, 50, 100, 200, and 500 pM / ml, and the final concentration of the internal standard peptide is 20 pM / ml. Take another 8 μl of 10 nM / ml extended internal standard working solution and add 392 μl of pure water to obtain a 200 pM / ml extended internal standard solution.

[0145] Optimized enzyme digestion method (trypsin)

[0146] Whole virus solutions from Jiangsu strain MDCK cell matrix and chicken embryo matrix were digested with enzymes at substrate to trypsin ratios of 10:1, 5:1, 2.5:1, 1.25:1, and 0.625:1, respectively. The condition for complete digestion was selected based on the peak area. After determining the optimal digestion ratio (enzyme:substrate = 1.25:1), digestion was performed at reaction times of 1, 2, 4, 8, and 16 hours. The optimal reaction time (2 hours) was selected based on the peak area.

[0147] Preprocessing methods

[0148] Based on the optimal conditions obtained from the above-mentioned enzyme digestion optimization method, the samples were pretreated. Take 10 μl of sample (protein concentration not exceeding 1 mg / ml), add 10 μl of 200 pM / ml extended internal standard solution and 20 μl of 0.2% Rapigest SF solution, mix well at 100℃ and heat for 5 min; after cooling, add 20 μl of 400 μg / ml trypsin solution, and digest in a 37℃ water bath for 2 hours; add 10 μl of 0.175 M hydrochloric acid solution, and incubate at room temperature for 30 minutes; add 30 μl of 0.1% formic acid solution, bringing the total volume to 100 μl; centrifuge at 13000 g for 10 min, and transfer the supernatant to a vial. Both Rapigest SF and trypsin were prepared using 50 mM ammonium bicarbonate solution and used immediately.

[0149] Liquid chromatography-mass spectrometry (LC-MS)

[0150] The liquid chromatography mobile phase A was 0.1% formic acid in water, and the mobile phase B was acetonitrile, with a flow rate of 0.3 ml / min. The gradient conditions were as follows: 0–6 min, mobile phase B increased from 5% to 60%; 6–6.5 min, increased from 60% to 95%; maintained for 0.5 min, then decreased from 95% to 5%; 7.01–10 min, maintained at the initial concentration of 5%. The column temperature was 40℃, and the autosampler temperature was 4℃.

[0151] Mass spectrometry was performed using an ESI ion source in positive ion mode of the MRM. The m / z and collision energy information of the ion pairs for the three peptides are shown in Table 2. The nebulizer flow rate was 3 L / min, the heater flow rate was 10 L / min, the interface temperature was 350℃, the DL temperature was 170℃, the heating block temperature was 400℃, and the dryer flow rate was 10 L / min.

[0152] Methodological Validation

[0153] Based on the prepared standard concentration points, the internal standard method was used to calibrate the curve (for details, please refer to: Cai Xuejian, Chen Feng, Yang Ling, et al. Application of internal standard method in ICP-MS [J]. Guangzhou Chemical Industry, 2015, 43(04):156-157+204.), to obtain the linear equation and R of the standard curve. 2 The detection limit (S / N=3) and quantitation limit (S / N=10) of the instrument were determined, and the detection limit and quantitation limit of the method were obtained based on a sample dilution factor of 10.

[0154] Two negative samples were used for injection to confirm the absence of interference in the target MRM channel and verify the specificity of the method. The repeatability of the method was verified through inter-day and intra-day precision. For inter-day precision, three parallel samples were prepared from four different samples, with each sample injected four times repeatedly. The concentration CV value for each sample was calculated between four independent experiments. For intra-day precision, six parallel samples were prepared from four different samples, with each sample injected four times repeatedly. The CV value for the concentration of each sample was calculated and should not exceed 20%.

[0155] Samples were analyzed using both the SRID method and the "SDS-PAGE + Lowry combination" method, and the results were compared with IDMS to verify accuracy. Since the standard antigen and antiserum reagents corresponding to the Jiangsu strain vaccine were not yet commercially available, SRID experiments could not be performed. Therefore, a method combining purity and protein content was used, and the results were compared with IDMS results to calculate the concordance rate. Simultaneously, the SRID method was used to compare the results of the Vietnamese strain vaccine, and the concordance rate was calculated to assess the closeness of the IDMS results to the true values; the results should be between 80% and 120%. The calculation formula is as follows.

[0156] Compliance rate =

[0157]

[0158] or =

[0159]

[0160] Example 2. Experimental Results

[0161] After theoretical enzyme digestion using software, four candidate sequences were selected: AIDGVTNK (SEQ ID NO: 1), TLDFHDSNVK (SEQ ID NO: 2), LVLATGLR (SEQ ID NO: 3), and EFNNLER (SEQ ID NO: 4). High-resolution mass spectrometry analysis of the digested vaccine samples yielded all four peptides. However, EFNNLER(R) exhibited a missed cleavage of one R group; therefore, EFNNLER was excluded from the candidate peptide pool.

[0162] The other three peptides are located at the HA1 terminus and HA2 of the HA sequence, respectively, and belong to relatively conserved regions (see [link to relevant documentation]). Figure 1 The isoleucine (I) of AIDGVTNK, the leucine (L) of TLDFHDSNVK, and the valine (V) of LVLATGLR were respectively... 12 C and 14 N replaced with stable isotopes 13 C and 15 N (For specific methods, please refer to: Villanueva J, Carrascal M, Abian J. Isotope dilution mass spectrometry for absolute quantification in proteomics: concepts and strategies. J Proteomics. 2014 Jan 16;96:184-99.), the molecular weight of the peptides was increased by 7, 6, and 7 Da, respectively, and isotopic internal standards with retention times consistent with the external standard peptides were synthesized. In addition, to simulate the enzymatic cleavage effect of real protein amino acid sequences, two amino acids were added to each side of the internal standard peptide according to the theoretical sequence to synthesize extended internal standard peptides, also known as extended peptides and winged peptides (for specific sequences, please refer to SEQ ID NO: 5 to SEQ ID NO: 7 in Table 1), which were processed together with the samples to reduce the influence of matrix effects.

[0163] Using peptide standard solutions of appropriate concentrations, ion pairs and ion source conditions, including collision energy, deflection voltage, DL temperature, and nozzle position, were determined and optimized on a triple quadrupole LC-MS / MS system. The mobile phase composition was adjusted, and the addition of 0.1% formic acid to mobile phases A and B was tested. The response rates of different combinations were compared, and the final determination was 0.1% formic acid in water for phase A and acetonitrile for phase B. The mobile phase gradient was adjusted to achieve peak elution within 5 minutes and equilibration to the initial concentration within 10 minutes (see [link to mobile phase analysis]). Figure 2 ; Figure 2 The peptide information is as follows: H5-1: AIDGVTNK; H5-4: TLDFHDSNVK; H5-7: LVLATGLR; H5-2: AIDGVTNK; H5-5: TLDFHDSNVK; H5-8: LVLATGLR), and the three peptides are well separated.

[0164] After instrument optimization, the R of the three peptides 2All values ​​were greater than 0.98, indicating good linearity. The limits of detection and quantitation of AIDGVTNK and LVLATGLR were significantly lower than those of the SRID method. The limit of quantitation of the SRID method was only 4 μg / ml (this result is recorded in the following literature on the detection of HA protein using the SRID method: Williams TL, Pirkle JL, Barr JR. Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry. Vaccine. 2012 Mar 23;30(14):2475-82. and Santana WI, Williams TL, Winne EK, Pirkle JL, Barr JR. Quantification of viral proteins of the avian H7 subtype of influenza virus: an isotopedilution mass spectrometry method applicable for producing more rapid vaccines in the case of an influenza pandemic. Anal Chem. 2014 May). 6;86(9):4088-95.), the IDMS method established in this application improves sensitivity by 6.7 times (see Table 3).

[0165] Table 3 Standard curves for specific peptides

[0166] Rapigest SF, an acid-labile ionic surfactant and protein denaturant, significantly enhances trypsin cleavage efficiency by dissolving and opening protein structures to expose internal cleavage sites. Based on the results of optimized cleavage conditions (see...),... Figure 3 When the enzyme-to-substrate ratio was 1.25:1, the peak areas of all three specific peptides were at their maximum. Optimizing the digestion time under this condition showed that most peptides reached a plateau within 2 hours, and the peak area no longer increased significantly. Therefore, this condition was chosen as the optimal method. Hydrochloric acid inactivates trypsin and degrades Rapidus SF, an acid-labile surfactant, thus avoiding interference from residual substances on the mass spectrometry results.

[0167] Negative samples from both matrices were tested, and the target MRM channels showed no corresponding responses, indicating good specificity of the method. The inter-day and intra-day precision of four different matrix samples were examined and compared with results from conventional methods. Sample 1 was a whole virus solution of the Vietnamese strain from MDCK cell matrix; Sample 2 was a lysate of the Vietnamese strain from MDCK cell matrix; Sample 3 was a whole virus solution of the Jiangsu strain from MDCK cell matrix; and Sample 4 was a whole virus solution of the Jiangsu strain from chicken embryo matrix. The concordance rates of Samples 1 and 2 were compared with SRID results, and the concordance rate of Sample 4 was compared with the combined results of SDS-PAGE and Lowry methods.

[0168] The experimental results showed that the intra-day and inter-day precision CVs of the peptide AIDGVTNK were 12–15% and 9–14%, respectively. Compared with the SRID results, the concordance rates were 85% and 101%, respectively. Compared with the SDS-PAGE and Lowry results, the concordance rate was approximately 86%. The precision and accuracy both met the requirements, indicating that the peptide has good repeatability and the detection results are reliable.

[0169] In comparison, the inter-day precision of TLDFHDSNVK and LVLATGLR were 23–57% and 35–46%, respectively, while the intra-day precision CVs were 17–22% and 21–34%, respectively, with CVs exceeding 20%, indicating poor reproducibility. The concordance rate of the TLDFHDSNVK peptide method was between 98% and 120%, while the concordance rate of the LVLATGLR peptide method was 102% to 591%, with the results for sample 1 being significantly higher, presumably due to inconsistent enzymatic digestion efficiencies of different peptides in that sample.

[0170] Table 4 IDMS Precision and Accuracy Data

[0171] To synthesize the methodological metrics of the three candidate peptides, the results showed that the AIDGVTNK peptide exhibited high sensitivity and good linearity (R0.05). 2 With a value >0.99, the precision and accuracy meet the requirements. This peptide is relatively conserved in vaccine strains such as Jiangsu strain and Vietnam strain, as well as popular strains such as A / Texas / 37 / 2024. It is the preferred HA quantitative peptide for H5N1 Jiangsu strain vaccine.

[0172] The peptide TLDFHDSNVK has relatively poor sensitivity but good linearity (R0). 2 >0.98), indicating poor intra-day precision but good intra-day precision and accuracy, making it a potential candidate for quantitative peptides; the LVLATGLR peptide exhibits high sensitivity and good linearity (RL). 2 (>0.99), but with the worst precision, it is considered for use as a qualitative peptide.

[0173] Compared with traditional immunological detection methods, the detection method proposed in this application has three major technical advantages: First, it eliminates the dependence on high-affinity and specific antibodies, significantly shortening the preparation cycle of immunized sheep from 2-3 months to 2-3 weeks; Second, the detection performance is comprehensively improved, with the linear range extended to 4 orders of magnitude (0.5-500 pM / ml), and the method's limit of quantitation is 6.7 times higher than that of SRID; Third, it adopts the MRM mode, which can simultaneously achieve accurate quantification of key proteins such as HA and NA, and there is no cross-interference between ion channels, exhibiting extremely strong specificity.

[0174] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A polypeptide having the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide of claim 1, wherein, The polypeptide has the amino acid sequence shown in SEQ ID NO: 1, and is used to determine the amount of H5N1 HA protein in a sample; Preferably, the polypeptide is unlabeled or has a detectable label; Preferably, the polypeptide is labeled with an isotope; Preferably, one or more (e.g., 1, 2, or 3) amino acid residues in the polypeptide are labeled with stable isotopes; Preferably, the stable isotope label is selected from... 2 H, 13 C 15 N、 17 O、 18 O, or any combination thereof; Preferably, the stable isotope labeling is as follows: 13 C and 15 N.

3. The polypeptide according to claim 1 or 2, wherein, The polypeptide has the following sequence: A[I]DGVTNK (SEQ ID NO: 1), where [I] indicates that one or more atoms in the isoleucine are replaced by their corresponding stable isotopes; Preferably, all carbon atoms in the isoleucine are... 13 C is replaced, and all nitrogen atoms are replaced. 15 N replacement; Preferably, the sample is an H5N1 cell matrix containing H5N1 HA protein (e.g., MDCK cell matrix) or a chicken embryo matrix vaccine virus solution.

4. A reagent kit comprising: A first polypeptide having the amino acid sequence shown in SEQ ID NO: 1, and the first polypeptide being unlabeled; and / or The second polypeptide has the same amino acid sequence as the first polypeptide and is labeled with an isotope. Preferably, the isotope label is as defined in claim 2 or 3.

5. The kit of claim 4, further comprising: The third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, and the third polypeptide is labeled with an isotope. Preferably, the third polypeptide further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids at both ends of the amino acid sequence shown in SEQ ID NO: 1, and is a truncated peptide of H5N1 HA protein; Preferably, the third polypeptide has the amino acid sequence shown in SEQ ID NO: 5; Preferably, the isotope label is as defined in claim 2 or 3.

6. The kit according to claim 4 or 5, wherein, The kit also contains reagents for mass spectrometry detection of the first polypeptide, the second polypeptide, and / or the third polypeptide; Preferably, the kit further comprises one or more reagents selected from 1)-5): 1) Denaturing agents (e.g., urea, guanidine hydrochloride, or ionic surfactants); 2) Reducing agents (e.g., dithiothreitol, mercaptoethanol, or TECP); 3) Alkylating agents (e.g., iodoacetamide); 4) Proteases (e.g., trypsin, LysC protease, GluC protease, AspN protease, or chymotrypsin); 5) Water (e.g., pure water, ultrapure water, deionized water, or distilled water); Preferably, the denaturant is Rapidus SF.

7. A method for determining the amount of H5N1 HA in a sample, comprising the step of determining the amount of a first polypeptide in the enzymatic digestion product of the sample using mass spectrometry, wherein, The first polypeptide has the amino acid sequence shown in SEQ ID NO: 1 and is unlabeled; Preferably, the amount of the first polypeptide in the enzyme digestion product of the sample is determined by isotope dilution mass spectrometry (IDMS); Preferably, the enzyme digestion product is a trypsin digestion product; Preferably, the sample is treated with a denaturing agent before enzymatic digestion; Preferably, before using mass spectrometry to determine the amount of the first polypeptide in the enzyme digestion product of the sample, the step of purifying the enzyme digestion product is further included; preferably, the purification is chromatography, such as liquid chromatography (e.g., high performance liquid chromatography or ultra-high performance liquid chromatography). Preferably, the sample is an H5N1 MDCK cell matrix containing H5N1 HA protein (e.g., MDCK cell matrix) or a chicken embryo matrix vaccine virus solution.

8. The method of claim 7, wherein, The method includes: (i) The sample is digested using a protease to obtain the enzyme digestion product; (ii) Perform mass spectrometry analysis on the enzyme digestion product obtained in step (i) and obtain the signal intensity of the first polypeptide; (iii) Compare the signal intensity obtained in step (ii) with a standard curve and obtain the molar content of the first polypeptide, which is the amount of H5N1 HA protein in the sample; wherein the standard curve is a linear relationship between the known amount of the first polypeptide and the signal intensity.

9. The method of claim 7, wherein, The method includes: (1) The sample was digested using a protease to obtain the enzyme digestion product; (2) Add a known amount of the second polypeptide to the enzyme digestion product obtained in step (1) and obtain a mixture; (3) Perform mass spectrometry analysis on the mixture obtained in step (2) and obtain the signal intensities of the first polypeptide and the second polypeptide; (4) Calculate the ratio of the signal intensity of the first peptide to that of the second peptide, and calculate the ratio of the concentration of the first peptide to that of the second peptide; (5) Compare the ratio with the standard curve and obtain the content of the first polypeptide, which is the amount of H5N1 HA protein in the sample; wherein, the standard curve is a linear relationship between the concentration ratio of the first polypeptide and the signal intensity ratio; Preferably, the protease is trypsin; Preferably, the signal strength is the peak height or peak area.

10. The method of claim 7, wherein, The method includes: (a) Providing a mixture comprising a known amount of a first polypeptide and a second polypeptide, performing mass spectrometry analysis on the mixture, and obtaining the signal intensities of the first polypeptide and the second polypeptide; wherein the second polypeptide has the same amino acid sequence as the first polypeptide and is labeled with an isotope; Then, based on the signal strength, a standard curve is plotted; (b) Providing a mixture comprising a known amount of a third polypeptide and a sample comprising H5N1 HA protein; wherein the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 and is labeled with an isotope; The mixture was digested using a protease to obtain an enzyme digestion product containing unknown amounts of a first polypeptide and a second polypeptide; the enzyme digestion product was then analyzed by mass spectrometry to obtain the signal intensity of the unknown amounts of the first polypeptide and the second polypeptide. (c) Compare the signal intensities of the first and second peptides obtained in step (b) with the standard curve, and analyze the content of the unknown amount of the first peptide, which is the amount of H5N1 HA protein in the sample. Steps (a) and (b) are not in any particular order.

11. The method of claim 10, wherein, In step (a), a linear regression analysis is performed using the peak area ratio of the first peptide and the second peptide and the corresponding concentration ratio of the first peptide and the second peptide to obtain a standard curve of the concentration of the first peptide relative to the peak area ratio (e.g., the first peptide or the second peptide). Then, the signal intensities of the first and second peptides obtained in step (b) are compared with the standard curve. Based on the amount of the third peptide before enzyme digestion and the amount of the second peptide generated after enzyme digestion, the content of the first peptide in the sample is calibrated, and the content of the unknown amount of the first peptide is analyzed. This content is the amount of H5N1 HA protein in the sample. Preferably, the sample is a cell matrix containing H5N1 HA protein (e.g., MDCK cell matrix) or a chicken embryo matrix vaccine virus solution; Preferably, in step (b), the ratio of the mixture to the protease is 2:1 to 1:1; for example, 2:1, 1.75:1, 1.5:1, 1.25:1 or 1:1; Preferably, in step (b), the digestion time is 1-4 hours; for example, 1 hour, 2 hours, 3 hours or 4 hours.

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