Specific antibody Fc segment glycosylation MRM quantitative analysis method and application thereof

Abstract

The invention discloses a specific antibody Fc segment glycosylation MRM quantitative analysis method and application thereof. The specific antibody Fc segment glycosylation MRM quantitative analysis method comprises the following steps: characterizing isotope labeled and non-isotope labeled glycopeptides and polypeptide standard substances; adding glycopeptide and polypeptide standard substances into the blank matrix to prepare standard solutions with different concentrations; the method comprises the following steps: performing pretreatment including antigen immobilization, immunoadsorption determination and protease hydrolysis splitting on a biological sample to be detected; according to the method, ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry is adopted for determination, and quantitative analysis of specific antibody subtypes and glycoforms is realized through characteristic ions of glycopeptides and polypeptide standard substances; and establishing a standard curve of glycopeptide and polypeptide standard substances containing the isotope internal standard, and calculating the antibody subtype and glycoform concentration of the biological sample to be detected. MRM quantitative analysis is carried out on patient serum specific antibody subtypes and glycoforms, the method is rapid, accurate and sensitive, and the fucose defect level and the galactose defect level have good clinical diagnosis value on virus infectious diseases and autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a method for quantitatively analyzing the glycosylation of the Fc segment of a specific antibody by MRM and its application. Background Art

[0002] Antibodies are central mediators of the immune system. IgG is the most important immunoglobulin. When the IgG / antigen complex binds to the membrane-bound Fcγ receptors (FcγRs), it triggers the immune response of cells, and this immune response is regulated by multiple factors. First, different IgG subclasses have different affinities for a given FcγR, and different FcγRs are heterogeneous in ligand specificity, expression pattern, and triggering effector functions. In addition, post-translational glycosylation modifications of antibodies and Fcγ receptors regulate the affinity of their interaction.

[0003] In the prior art, a number of studies have shown that the lack of fucose at the Fc end of human IgG antibodies plays a key role in enhancing antibody-dependent cell cytotoxicity. Shields et al. revealed the influence of fucosylated oligosaccharides on antibody effector functions, including binding to human FcγR, C1q, FcRn, and ADCC. The binding of fucose-deficient IgG1s to FcγRIIIa increased by 50-fold and enhanced the ADCC effect. Toyohide Shinkawa et al. found that using purified human peripheral blood mononuclear cells as effectors, the antibody-dependent cell cytotoxicity (ADCC) of anti-human interleukin 5 receptor (hIL-5R), humanized immunoglobulin G1 (IgG1), and anti-CD2 chimeric IgG0 produced by the rat hybridoma YB20 / 1 cell line was more than 50 times higher than that produced by the Chinese hamster ovary (CHO) cell line.

[0004] Under normal circumstances, IgG in human plasma is highly fucosylated, and afucosylated antigen-specific IgG responses have played important roles in various pathological processes, including alloimmune responses to blood cells, the immune response to Plasmodium falciparum antigens expressed on red blood cells, and the immune response to viruses such as human immunodeficiency virus, dengue virus, and SARS-CoV-2. Regarding the main mechanism of action of Fc-core fucosylated IgG1 antibodies, IgG1 antibodies lacking core fucose bind to FcɣRIIIA and FcɣRIIIB on the indicated immune cells (NK, Mø, and PMN) 10-100 times more strongly, which leads to an increase in ADCC by NK cells, enhanced competition with plasma IgG, increased PMN activation, and increased inhibition of FcγRIIA-mediated ADCC by PMN. In addition, in a variety of autoantibody-dependent and autoantibody-independent autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and inflammatory bowel disease, changes in antibody glycosylation have also been observed during active disease, and the most common is an increase in the IgG-G0F glycoform lacking terminal galactose and sialic acid residues during the inflammatory phase.

[0005] In patients with severe novel coronavirus infection, the inhibition of B cell FUT8 glycosyltransferase leads to a significant increase in the level of afucosylated IgG antibodies, and afucosylation causes a stronger immune response, which in turn triggers a cytokine storm and acute respiratory distress syndrome, ultimately resulting in the development of severe COVID-19. Severe COVID-19 is characterized by inflammatory features, including high levels of inflammatory cytokines, alveolar inflammatory infiltration, and vascular microthrombi. Severe COVID-19 patients exhibit unique serological features, including increased IgG1 with afucosylated Fc glycans. This Fc modification of SARS-CoV-2 IgG enhances the interaction with and activation of FcγR, FcγRIIIa. When incorporated into immune complexes, fucose-deficient Fc receptors enhance the production of inflammatory cytokines by monocytes (including IL-6 and TNF).

[0006] The variable region of the immunoglobulin IgG molecule directly exerts neutralizing and blocking effects by binding to viral envelope proteins. The crystallizable fragment (Fc) region of the IgG molecule can bind to complement and also to immune cells expressing Fc receptors. Immune cells expressing Fc receptors include monocytes, macrophages, NK cells, etc. After the antibody binds to immune cells through the Fc segment, it can regulate the functions of immune cells. Fc receptors are divided into two categories: activating and inhibitory. The N-glycosylation sites in the Fc segment have complex and diverse N-glycan structural modifications, among which core fucose is one of the important modifications. These N-glycans can regulate the affinity of Fc for binding to Fc receptors. For example, the core fucose of N-glycans regulates the binding of Fc to the activating Fc receptor (FcR IIIa). After the N-glycans are modified by core fucose, the affinity for FcRIIIa decreases by dozens of times, which is a molecular mechanism to avoid overactivation of FcRIIIa.

[0007] The Jeffery Ravetch research group at Rockefeller University found that the affinity between IgG1 lacking core fucose and FcR IIIa increased, leading to severe dengue fever. After the outbreak of the novel coronavirus infection, the Wang lab at Stanford University used the Biolayer Interferometry (BLI) method to detect the affinity between RBD-specific IgG in severe infections and FcRIIIa, and found that compared with the mild infection group, the binding of RBD-specific IgG to recombinant Fc RIIIa in the severe infection group was significantly enhanced. In the experiment of activating monocytes, RBD-specific IgG in the severe infection group caused the release of cytokines from monocytes, while RBD-specific IgG in mild patients could not activate monocytes to release cytokines. At the same time as the Taia Wang team reported the above findings, Gestur Vidarsson at the University of Amsterdam also reported in Science the fucose deficiency of anti-spike protein (S protein) IgG in severe infections.

[0008] Studies have shown that in experimental and human autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and infections), abnormal IgG glycosylation (absence of galactose and sialic acid) is significantly increased. The team of Jörg Köhl at the University of Lübeck found that the high galactosylation of the N-glycans of IgG1 molecules forms an inhibitory pathway by promoting the co-signaling of FcγRIIB and dectin-1, blocking the pro-inflammatory functions of the C5aR and CXCR2 chemokine receptors. The team of Jeffery Ravetch revealed that in inflammatory and autoimmune states, immune complexes trigger the activation of inflammatory macrophages through activating FcRs, while highly sialylated Fc induces the secretion of anti-inflammatory mediators by binding to the SIGN-R1 lectin of regulatory macrophages in the marginal zone of the spleen. These mediators act on effector macrophages at the lesion site, upregulating the expression of inhibitory FcgRIIB, thereby increasing the activation threshold of immune complexes and ultimately reducing the inflammatory damage mediated by autoantibodies.

[0009] In summary, in viral infectious diseases and some autoimmune diseases, the differences in the subtypes and glycosylation modifications of specific antibodies have important effects on the occurrence and development of diseases. After the novel coronavirus, influenza virus, and RSV virus vaccines have been successively put into use globally, the subtypes and fucosylation modifications of virus-specific antibodies are still unclear. Especially in the elderly population and those with underlying diseases, the deficiencies of Th1 cell-induced antibody subtypes (IgG1 and IgG3 subtypes) and fucosylated antibodies are potential risks for reducing vaccine efficacy. Multiple laboratories have carried out rapid detection and analysis studies on specific antibodies of the novel coronavirus vaccine and influenza vaccine, but there is still a lack of standardized analytical methods that can be promoted and used by third-party laboratories. Therefore, while optimizing antibody research and treatment, it is necessary to pay attention to and strengthen the research on the quantitative analysis of antibody Fc glycosylation to achieve real-time monitoring and prevention effects on the occurrence of severe diseases. Summary of the Invention

[0010] The main object of the present invention is to provide a method for quantitative analysis of multiple reaction monitoring (MRM) of Fc glycosylation of specific antibodies. By synthesizing characteristic peptide standards of each IgG subtype and 6 glycopeptide standards of the Fc segment: G0F, G0NF, G1, G1N, G2, G2S, using an ultra-high performance liquid chromatograph in combination with an SCIEX 7500 triple quadrupole mass spectrometer, an MRM quantitative analysis method suitable for characteristic polypeptides and glycopeptides of antibodies is established, and a set of standard curves containing internal standards is constructed for quantitative analysis.

[0011] Another object of the present invention is to provide an application of the method for quantitatively analyzing the glycosylation of the Fc segment of the specific antibody by MRM, including quantitatively determining the absolute content of the specific antibody in the serum of patients infected with the novel coronavirus and the absolute quantification of 6 glycoforms of the Fc segment, instantaneously monitoring the fucose deficiency and galactose deficiency of the specific antibody of the patient, and indicating the severity of the disease according to the degree of glycoform deficiency, so as to achieve timely prevention and monitoring effects.

[0012] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a method for characterizing a glycopeptide and a characteristic polypeptide standard, and the method includes the following steps: (1) Synthesizing isotopically labeled and non-isotopically labeled glycopeptides and characteristic polypeptide standards; (2) Characterizing the synthesized isotopically labeled glycopeptides and characteristic polypeptide standards; (3) Characterizing the synthesized non-isotopically labeled glycopeptides and characteristic polypeptide standards; (4) Constructing a liquid chromatography-mass spectrometry characterization by using a combination of an ultra-high performance liquid chromatograph and a triple quadrupole mass spectrometer.

[0013] In the second aspect, the present invention provides a method for quantitatively analyzing the glycosylation of the Fc segment of a specific antibody by MRM, and the method includes the following steps: (i) Setting MRM parameters by using a combination of an ultra-high performance liquid chromatograph and a triple quadrupole mass spectrometer; (ii) Constructing an IgG1-GPSVFPLAPSSK standard curve containing an isotopic internal standard based on the MRM parameter setting; (iii) Constructing a mixed standard curve of 6 glycopeptides containing an isotopic internal standard including G0F, G0NF, G1, G1N, G2, and G2S based on the MRM parameter setting.

[0014] Preferably, in step (ii), the concentration gradient of the IgG1-GPSVFPLAPSSK standard curve is set to 5 ng / μL, 1 ng / μL, 500 pg / μL, 100 pg / μL, 20 pg / μL, and the internal standard concentration is set to 1 ng / μL.

[0015] Preferably, in step (iii), the concentration gradient of the mixed standard curve of the 6 glycopeptides is set to 500 pg / μL, 200 pg / μL, 100 pg / μL, 50 pg / μL, 10 pg / μL, 2 pg / μL, and the internal standard concentration is set to 100 pg / μL.

[0016] In the third aspect, the present invention provides a pretreatment method for a patient serum sample, and the method includes: (a)Immobilization of disease antigen of the patient to be detected, immunosorbent assay, proteolytic cleavage; (b)Based on the synthesized isotopically labeled glycopeptides and characteristic polypeptide standards obtained by the characterization method, and the standard curve containing isotope internal standards obtained by the specific antibody Fc glycosylation MRM quantitative analysis method, absolute quantification of the specific antibody subtypes and glycopeptide MRM in the patient serum sample is performed.

[0017] Preferably, the diseases include novel coronavirus, influenza virus and RSV virus.

[0018] The present invention synthesizes six glycopeptides of the characteristic peptides and Fc segments of isotopically labeled IgG subtypes: G0F, G0NF, G1, G1N, G2, G2S. The synthesized isotopic standards are used as internal standards for the standard curve to correct instrument response fluctuations and matrix effects, improve quantitative accuracy, and at the same time improve the sensitivity and specificity of detection, providing a powerful tool for clinical diagnosis.

[0019] Compared with the prior art, the beneficial effects of the present invention are at least as follows: 1. High sensitivity and accuracy: By combining an ultra-high performance liquid chromatograph Exion LC AD with an SCIEX 7500 triple quadrupole mass spectrometer and combining with the MRM method, a quantitative analysis technique with high sensitivity and accuracy is proposed, which helps to accurately detect and quantify target polypeptides and glycopeptides in complex biological samples.

[0020] 2. Use of isotope internal standards: The present invention uses isotope internal standards to establish standard curves for characteristic polypeptides and glycopeptides, which helps to correct matrix effects, instrument fluctuations and losses during sample preparation in the analysis process, and further improves the accuracy and repeatability of quantitative analysis.

[0021] 3. Strong specificity: The present invention performs quantitative analysis on the specific antibody subtypes and glycan deficiencies in the serum of patients infected with novel coronavirus, especially in the detection of fucose and galactose deficiency levels, providing new biomarkers for the diagnosis of viral infectious diseases and autoimmune diseases.

[0022] 4. Clinical diagnostic value: The present invention provides valuable information for the clinical diagnosis of viral infectious diseases and autoimmune diseases by detecting fucose deficiency and galactose deficiency levels, which helps in the early detection, treatment monitoring and prognosis evaluation of diseases.

[0023] 5. Fast, accurate and sensitive analysis: The present invention combines the MRM quantitative method of liquid chromatography-mass spectrometry and the specificity of immunosorption, and can quickly, accurately and sensitively analyze the content of disease-related specific antibodies and glycan deficiency levels, which is of great significance for clinical diagnosis and patient management.

[0024] 6. Methodological innovation: The present invention provides a new methodology that applies the technology of liquid chromatography-mass spectrometry coupling to the precise quantitative analysis of serum-specific antibody subtypes and glycan deficiency, filling the gap in the existing technology to a certain extent.

[0025] 7. Wide application prospects: The present invention is not only applicable to the analysis of serum from patients infected with the novel coronavirus, but can also be extended to the research and diagnosis of other diseases, with broad application prospects and potential. Description of the drawings

[0026] Figure 1 Schematic diagram of the MRM quantitative analysis method for antibody Fc segment glycosylation and its application in the examples.

[0027] Figure 2 Schematic diagram of the characterization of 6 glycopeptide standards labeled with isotopes in the examples.

[0028] Figure 3 Total MRM chromatograms of the standards and samples to be measured in the examples.

[0029] Figure 4 Schematic diagram of the basic structure of glycopeptides in the examples; a) Composition of the oligosaccharide part: blue squares represent N-acetylglucosamine, green circles represent mannose, red triangles represent fucose, yellow circles represent galactose, and purple quadrilaterals represent sialic acid; b) The peptide segment is EEQYNSTYR.

[0030] Figure 5 Overall standard curve of 6 glycopeptide standards containing isotope internal standards in the examples.

[0031] Figure 6 Standard curve of the characteristic polypeptide standard of IgG1 containing isotope internal standard in the examples.

[0032] Figure 7 Virus-specific antibody concentrations in patients infected with the novel coronavirus and influenza virus in the examples.

[0033] Figure 8 Percentage of glycan deficiency at the Fc end of virus-specific antibodies in patients infected with the novel coronavirus and influenza virus in the examples (S-309 is a standard antibody expressed in 293T cells).

[0034] Specific examination method for substantive examination

[0035] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below in conjunction with specific embodiments and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. It should be noted that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all fall within the protection scope of the present invention.

[0036] The following embodiments use an ultra-high performance liquid chromatograph Exion LC AD (AB SCIEX LLC, USA) and an SCIEX 7500 triple quadrupole mass spectrometer (AB SCIEX LLC, USA).

[0037] The following embodiments propose a method for quantitative analysis of the glycosylation of the Fc segment of a specific antibody by MRM, including the following steps: (1) Synthesize isotope-labeled and non-isotope-labeled glycopeptide standards and polypeptide standards; (2) Characterize the isotope-labeled and non-isotope-labeled glycopeptide standards and polypeptide standards; (3) Add glycopeptide standards and polypeptide standards to a blank matrix to prepare standard solutions of different concentrations; (4) Pretreat the biological sample to be tested, including: antigen immobilization, immunoassay, and protease hydrolysis and cleavage; (5) Use ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry for determination, and perform quantitative analysis of specific antibody subtypes and glycoforms with the characteristic ions of glycopeptides and polypeptides; (6) Establish standard curves of glycopeptides and polypeptides containing isotope internal standards, and calculate the concentrations of antibody subtypes and glycoforms in the biological sample to be tested.

[0038] In some embodiments, the biological sample to be tested is plasma, serum, or a test solution containing a biological matrix.

[0039] In some embodiments, when the biological sample to be tested is plasma or serum, the pretreatment in step (4) is: immobilize the corresponding antigen protein of the sample to be tested with an antigen-coated plate; after immobilizing the antigen protein, add the diluted serum sample to be tested for immunosorption; after adsorption, take the eluate, concentrate and dry it; add trypsin solution to the dried sample for reaction, and vacuum dry after the reaction ends.

[0040] In some embodiments, an internal standard method or an external standard method is used for quantitative determination.

[0041] In some embodiments, in step (5), the quantitative analysis includes the analysis of serum specific antibody subtypes and glycoforms of viral infectious diseases and / or autoimmune diseases caused by novel coronavirus, influenza virus, and RSV virus. The proportion of fucose deficiency is calculated by normalizing the sum of the signal intensities of fucose-deficient glycoforms (G1, G1N, G2, G2S) to the intensity of the IgG1-GPSVFPLAPSSK peptide. The proportion of galactose deficiency is calculated by normalizing the sum of the signal intensities of galactose-deficient glycoforms (G0F, G0NF) to the intensity of the IgG1-GPSVFPLAPSSK peptide.

[0042] In some embodiments, in step (5), the liquid chromatography conditions include: an ultra-high performance liquid chromatography system; a reverse-phase chromatography column; a water / acetonitrile mobile phase; and gradient elution.

[0043] In some embodiments, in step (5), the liquid chromatography conditions include: an Acquity PREMIER Peptide BEHC18 chromatographic column; column temperature: 40 °C; the mobile phase includes: phase A: 0.1% formic acid-aqueous solution and phase B: 0.1% formic acid-acetonitrile solution; flow rate: 0.3 mL / min; gradient elution is carried out under the following conditions: the gradient starts from 2% of mobile phase B, is held for 0.5 min, then at 9.0 min, the percentage of phase B is increased to 38%, then at 10.5 min, it is increased to 95%, and is held until 12.5 min; finally, at 12.6 min, the percentage of phase B drops to 2% and is held for 2.4 min, and the total running time is 15 min.

[0044] In some embodiments, in step (5), the triple quadrupole mass spectrometry conditions include: an ESI ion source, positive ion mode, and MRM detection mode.

[0045] In the following embodiments, the above-mentioned MRM quantitative analysis method for the glycosylation of the Fc segment of specific antibodies is also applied to the analysis of serum specific antibody subtypes and glycoforms of viral infectious diseases and / or autoimmune diseases caused by novel coronavirus, influenza virus, and RSV virus. Example

[0046] 1. Characterization of standards 1.1 Information on synthetic isotope-labeled and non-isotope-labeled glycopeptides (G0F, G0NF, G1, G1N, G2, G2S) and polypeptides The isotope-labeled glycopeptides and polypeptides were characterized by a high-resolution ZenoTOF 7600 System. The isotope-labeling positions of the glycopeptides are 13 C6, 15 N4]-Arginine, and the isotope-labeling positions of the polypeptides are 13 C6,15 N4]-Arginine and 13 C6, 15 N2]-Lysine, as shown in Table 1 and Figure 3 as follows.

[0047] Table 1: Information Table of Glycopeptide and Polypeptide Standards Serial number type name 1 Glycopeptides G0F-EEQYNSTYR 2 Glycopeptides G1-EEQYNSTYR 3 Glycopeptides G2-EEQYNSTYR 4 Glycopeptides G0NF-EEQYNSTYR 5 Glycopeptides G1N-EEQYNSTYR 6 Glycopeptides G2S-EEQYNSTYR 7 Isotope-labeled glycopeptides G0F-EEQYNSTYR* 8 Isotope-labeled glycopeptides G1-EEQYNSTYR* 9 Isotope-labeled glycopeptides G2-EEQYNSTYR* 10 Isotope-labeled glycopeptides G0NF-EEQYNSTYR* 11 Isotope-labeled glycopeptides G1N-EEQYNSTYR* 12 Isotope-labeled glycopeptides G2S-EEQYNSTYR* 13 IgG1 characteristic peptide GPSVFPLAPSSK 14 IgG2 characteristic peptide GLPAPIEK 15 IgG3 characteristic peptide WYVDGVEVHNAK 16 IgG4 characteristic peptide TTPPVLDSDGSFFLYSR 17 IgG characteristic peptide DTLMISR 18 Isotope-labeled peptides GPSVFPLAPSSK* 19 Isotope-labeled peptides GLPAPIEK* 20 Isotope-labeled peptides WYVDGVEVHNAK* 21 Isotope-labeled peptides TTPPVLDSDGSFFLYSR* 22 Isotope-labeled peptides DTLMISR* 1.2 Characterization of Synthesized Isotope-Labeled and Non-Isotope-Labeled Glycopeptides and Polypeptides The liquid phase and mass spectrometry methods for characterization are as follows: a. Liquid phase method Use the SCIEX ExionLC™ AC liquid phase system, the chromatographic column is Kinetex 2.6 μm C18 100 Å, LCColumn 100×2.6 mm. Mobile phase A: 2% acetonitrile / water, 0.1% formic acid; Mobile phase B: 98% acetonitrile / water, 0.1% formic acid. The flow rate is 0.3 mL / min, column temperature: 45 °C; injection volume: 1 μL; separation gradient: see Table 2.

[0048] Table 2: Liquid Phase Gradient Time(min) Flow (mL / min) B.Conc(%) B.Curve 0.00 0.3 2 0 0.50 0.3 2 0 9.00 0.3 38 0 9.50 0.3 95 0 11.50 0.3 95 0 11.60 0.3 2 0 b. Mass spectrometry method Use the SCIEX Zeno TOF 7600 mass spectrometry system for data acquisition, and the source gas parameters are as follows: spray gas: 55 psi, auxiliary heating gas: 55 psi, curtain gas: 35 psi, ion source temperature: 500 °C, ion source voltage: 5500 V, collision gas: 7 psi, declustering voltage: 60 V. Acquisition mode: information-dependent acquisition (IDA), primary scan range 400 - 2500, primary accumulation time 0.25 s, Time bins to sum set to 8; secondary scan range 100 - 3000, secondary accumulation time 0.1 s, Time bins to sum set to 8; collision energy: dynamic collision energy.

[0049] c. Based on the above liquid chromatography - mass spectrometry method, the characterization results are shown in Table 3.

[0050] Table 3: Characterization Results of Glycopeptide and Polypeptide Standards Standards Theoretical average molecular weight Characterization of molecular weight G0F 2634.55 2634.3 G1 2650.54 2650.4 G2 2812.69 2812.5 G0NF 2837.74 2837.1 G1N 2853.74 2853.1 G2S 3103.94 3103.2 IS_G0F 2643.91 2644.2 IS_G1 2659.90 2660.2 IS_G2 2821.96 2822.3 IS_G0NF 2846.90 2847.3 IS_G1N 2862.98 2863.3 IS_G2S 3113.81 3113.5 IgG1_GPSVFPLAPSSK 1186.34 1186.5 IgG2_GLPAPIEK 823.98 824.0 IgG3_WYVDGVEVHNAK 1416.55 1416.7 IgG4_TTPPVLDSDGSFFLYSR 1902.04 1902.0 IgG_DTLMISR 834.97 834.9 IS_IgG1_GPSVFPLAPSSK 1194.34 1194.4 IS_IgG2_GLPAPIEK 831.98 831.9 IS_IgG3_WYVDGVEVHNAK 1424.55 1424.5 IS_IgG4_TTPPVLDSDGSFFLYSR 1912.04 1912.0 IS_IgG_DTLMISR 844.97 844.8 2. Construction of Standard Curves Containing Isotope Internal Standards 2.1 Construction of a Mixed Standard Curve of IgG1 - GPSVFPLAPSSK Containing Isotope Internal Standard and 6 Glycopeptides: a. Prepare the loading buffer: 2% CAN (0.1% HA). The standard curve is in a 100 μL system. The concentration of IgG1 - GPSVFPLAPSSK with isotope internal standard is set to 1 μg / mL, and the concentration of glycopeptide with isotope internal standard is set to 100 ng / mL. Inject 2 μL for sample injection.

[0051] b. Prepare the internal standard mixture system (IS - MIX): 10 μL of 10 μg / mL glycopeptides (G0F, G0NF, G1, G1N, G2, G2S) + 10 μL of 100 μg / mL IgG1 - GPSVFPLAPSSK.

[0052] c. Prepare each concentration point of the standard curve as shown in Table 4: Table 4: Preparation of the standard curve

[0053] 2.2 MRM Liquid Chromatography and Mass Spectrometry Methods Use an ultra - high - performance liquid chromatograph Exion LC AD (AB SCIEX LLC, USA) coupled with an SCIEX 7500 triple quadrupole mass spectrometer (AB SCIEX LLC, USA). Chromatographic separation is carried out using an Acquity PREMIER Peptide CSH C18 column (2.1x100 mm, 1.7 μm, Waters). Mobile phase A is water containing 0.1% formic acid, and mobile phase B is acetonitrile containing 0.1% formic acid. For the analysis of glycopeptides, the gradient starts from 2% of mobile phase B, holds for 0.5 min, then at 9.0 min, the percentage of mobile phase B increases to 38%; then, at 10.5 min, it increases to 95% and holds until 12.5 min; finally, at 12.6 min, the percentage of mobile phase B drops to 2% and holds for 2.4 min. The total running time is 15 min, the column temperature is 40 °C, and the flow rate is 0.3 mL / min.

[0054] The SCIEX triple QuadTM 7500 mass spectrometer is equipped with an electrospray OptiFlow Pro ion source for positive multiple reaction monitoring (MRM) quantification. The MS analysis conditions are as follows: The turbo temperature is set to 450 °C, and the gas values are set as follows: curtain gas 40 psi, GS1 gas 50 psi, GS2 gas 50 psi, and the collision - activated dissociation (CAD) gas pressure is set to 10 psi. Other parameters are ion spray voltage (1.7 kV), dwell time (5 ms). All analyte - specific MRM parameters and m / z ratios are shown in Table 5.

[0055] Table 5: MRM parameters for monitoring glycopeptides (HexNAc)4(Hex)3(Fuc)1 G0F 878.69 204.1 15 10 19 11.5 (HexNAc)4(Hex)4 G1 884.021 204.1 15 10 17 11.5 (HexNAc)4(Hex)5 G2 938.039 366.1 15 10 17 11.5 (HexNAc)5(Hex)3(Fuc)1 G0NF 946.384 204.1 15 10 23 11.5 (HexNAc)5(Hex)4 G1N 951.715 204.1 15 10 30 11.5 (HexNAc)4(Hex)5(NeuAc)1 G2S 1035.071 366.1 15 10 39 11.5 (HexNAc)4(Hex)3(Fuc)1 IS_G0F 882.0 204.1 15 10 19 11.5 (HexNAc)4(Hex)4 IS_G1 887.4 204.1 15 10 17 11.5 (HexNAc)4(Hex)5 IS_G2 941.4 366.1 15 10 17 11.5 (HexNAc)5(Hex)3(Fuc)1 IS_G0NF 949.7 204.1 15 10 23 11.5 (HexNAc)5(Hex)4 IS_G1N 955.1 204.1 15 10 30 11.5 (HexNAc)4(Hex)5(NeuAc)1 IS_G2S 1038.4 366.1 15 10 39 11.5 IgG1_GPSVFPLAPSSK GPSVFPLAPSSK 593.8 699.4 15 10 28.1 11.5 IgG2_GLPAPIEK GLPAPIEK 412.8 327.7 15 10 19.2 11.5 IgG3_WYVDGVEVHNAK WYVDGVEVHNAK 708.9 469.3 15 10 33.9 11.5 IgG4_TTPPVLDSDGSFFLYSR TTPPVLDSDGSFFLYSR 951.5 850.4 15 10 45.9 11.5 IgG_DTLMISR DTLMISR 418.2 506.3 15 10 19.5 11.5 IgG1_GPSVFPLAPSSK IS_GPSVFPLAPSSK 597.8 707.4 15 10 28.3 11.5 IgG2_GLPAPIEK IS_GLPAPIEK 416.8 331.7 15 10 19.4 11.5 IgG3_WYVDGVEVHNAK IS_WYVDGVEVHNAK 712.9 477.3 15 10 33.9 11.5 IgG4_TTPPVLDSDGSFFLYSR IS_TTPPVLDSDGSFFLYSR 956.5 855.4 15 10 46.9 11.5 IgG_DTLMISR IS_DTLMISR 423.2 516.3 15 10 19.7 11.5 BSA LGEYGFQNALIVR 740.4 813.49 15 10 15 11.5 EP: entrance potential; CE: collision energy; CXP: collision exit potential.

[0056] 3. Pretreatment Preparation of Patient Serum Samples 3.1. Antigen Immobilization: a. Dilute Recombinant 2019 - nCoV S - trimer Protein (novoprotein, Cat. No.: DRA49) to 2 μg / mL in phosphate - buffered saline (PBS; pH 7.4), mix the tetravalent influenza protein antigens (H1N1, BY, H3N2, and BV subtypes; Chengdu Oulin Biologics) in equimolar ratio, and dilute to a total protein concentration of 2 μg / mL with phosphate - buffered saline (PBS; pH 7.4).

[0057] b. Transfer the diluted antigen into the wells of a 96 - well plate, 200 μL / well, seal the plate and incubate at 37 °C for 3 h to ensure antigen coating on the plate.

[0058] c. Wash the plate: Remove the protein solution and wash three times with 250 μL PBS - T for 2 min each time.

[0059] 3.2. Immunosorbent Assay: a. 20 μL serum + 180 μL PBS - T, 10 - fold dilution, 200 μL / well, incubate overnight at 4 °C to capture IgG.

[0060] b. Wash the plate: Wash each well 3 times with 250 μL PBS - T, then wash 2 times with 250 μL PBS, and wash 2 times with 250 μL 50 mM ABC for 2 min each time.

[0061] c. Add 200 μL 100 mM formic acid to each well with a multichannel pipette and incubate for 5 min with shaking at room temperature (300 rpm) to elute IgG, transfer the eluate to a 0.5 mL EP tube (Eppendorf Protein LoBind® Tubes (0030108094)).

[0062] d. Dry the eluate in a centrifugal vacuum dryer speed vac at 50 - 60 °C for 4 - 6 h.

[0063] 3.3 Protease Hydrolysis Cleavage a. Resuspend the dried IgG in 20 μL of 50 mM ABC for 5 minutes, and shake at 450 rpm at room temperature.

[0064] b. Dilute the acidic trypsin solution to 50 ng / μL in ice-cold water at a concentration of 1:20 (volume ratio) to obtain a trypsin cleavage mixture. Add 20 μL of the trypsin cleavage mixture to each well, and shake at 450 rpm and room temperature for 5 minutes.

[0065] c. Seal with a sticky sealing plate and incubate overnight at 37 °C (recommended incubation time: 18 - 22 h).

[0066] d. Dry the eluate in a centrifugal vacuum dryer speed vac at 37 °C for 4 - 6 hours, and store it at -80 °C after drying.

[0067] 3.4 LC-MS measurement Before loading onto the machine, resuspend the dried digested sample in 10 μL of loading buffer (2% ACN (0.1% HA), containing isotopic internal standards of 6 glycopeptides and characteristic polypeptides) for 5 minutes, shake at 450 rpm at room temperature, centrifuge the sample at 780 g for 10 minutes at room temperature, and store the sample in the thermostatic autosampler of the LC-MS instrument at 4 °C until injection.

[0068] 4. Result analysis For the specific antibody subtypes of patient serum samples and the absolute quantification of each glycopeptide MRM, absolute quantification of the samples was performed using the constructed standard curve, and the percentages of fucose and galactose deficiency were analyzed, as shown in Table 6 Figure 7 and Figure 8 as shown. The results showed that the average concentration of Spike-specific IgG1 in SARS-CoV2-infected patients (2 months after infection) was 7.65 μg / mL (51.00 nM), and the average concentration of hemagglutinin-specific IgG1 in influenza virus-infected patients was 5.37 μg / mL (35.80 nM). Analysis of influenza and COVID-19-infected patients using the detection method based on the calibration curve revealed significant glycan differences: the level of core fucosylation in influenza-infected patients was significantly reduced (22%), while the level of galactosylation increased in both disease groups.

[0069] In addition, the second and third points of the standard curve concentration were repeated 3 times for loading onto the machine, and 3 samples were randomly selected from the patient samples and repeated 3 times for loading onto the machine for detection. As shown in Table 7, the coefficient of variation of the repeated injection samples was analyzed to be within 10%, indicating high precision of the detection system, high consistency in sample processing and preparation, and strong data reliability.

[0070] Table 6: Serum specific antibodies, molar amounts of each glycopeptide, and levels of glycan defects in samples from influenza and novel coronavirus-infected patients calculated based on the standard curve Note: Fucose defect (%): [(G1 + G1N + G2 + G2S) / GPS]; Galactose defect (%): [(G0F + G0NF) / GPS] GPS: GPSVFPLAPSSK; jyc, gl, ywh, wn, jj, hy are samples from influenza patients; A11, A10, A13, A8, D1, D13, D3, D11, D6, D2 are samples from novel coronavirus-infected patients; s309 is a standard antibody with complete core fucose expressed by 293T cells.

[0071] Table 7: Statistics of coefficient of variation for samples and standard curves sample IgG1-GPS G0F G0NF) G1 G1N G2 G2S jyc 2.54% 2.54% 3.25% 4.00% 0.61% 6.37% 0.94% A11 4.32% 4.32% 7.66% 1.81% 4.95% 9.81% 4.40% D1 2.10% 2.10% 2.35% 9.88% 6.15% 3.00% 2.77% gs1 - 6.3% 8.8% 3.2% 5.9% 8.5% 8.3% gs2 - 5.2% 2.2% 2.6% 5.5% 1.3% 9.3% ps1 5.2% - - - - - - ps2 2.8% - - - - - - Note: gs1: glycopeptide-standard1 (100 ng / mL); gs2: glycopeptide-standard1 (50 ng / mL); ps1: peptide standard1 (1 μg / mL); ps2: peptide standard1 (0.5 μg / mL).

[0072] The usage conditions of the present invention: The antibody concentration against a specific antigen reaches 1 μg / mL.

[0073] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for MRM quantitative analysis of the glycosylation of the Fc segment of a specific antibody, characterized in that, It includes the following steps: (1) Synthesize isotopically labeled and non-isotopically labeled glycopeptide standards and polypeptide standards; (2) Characterize the isotopically labeled and non-isotopically labeled glycopeptide standards and polypeptide standards; (3) Add glycopeptide standards and polypeptide standards to a blank matrix to prepare standard solutions with different concentrations; (4) Pretreat the biological sample to be tested, including: antigen immobilization, immunosorbent assay, protease hydrolysis and cleavage; (5) Use ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry for determination, and perform quantitative analysis of specific antibody subtypes and glycoforms with the characteristic ions of glycopeptides and polypeptides; (6) Establish a standard curve for glycopeptide standards and polypeptide standards containing isotope internal standards, and calculate the concentrations of antibody subtypes and glycoforms in the biological sample to be tested.

2. The specific antibody Fc segment glycosylation MRM quantitative analysis method according to claim 1, characterized in that The biological sample to be tested is plasma, serum or a test solution containing a biological matrix.

3. The specific antibody Fc segment glycosylation MRM quantitative analysis method according to claim 2, characterized in that, When the biological sample to be tested is plasma or serum, the pretreatment in step (4) is: immobilize the corresponding antigen protein of the test sample with an antigen-coated plate; after immobilizing the antigen protein, add the diluted test serum sample for immunosorption; after adsorption, take the eluate and concentrate and dry it; add trypsin solution to the dried sample for reaction, and after the reaction ends, dry it under vacuum.

4. The MRM quantitative analysis method for the glycosylation of the Fc segment of the specific antibody according to claim 1, wherein Quantitative determination is carried out by the internal standard method or the external standard method.

5. The MRM quantitative analysis method for the glycosylation of the Fc segment of the specific antibody according to claim 1, wherein In step (5), the quantitative analysis includes the analysis of serum specific antibody subtypes and glycoforms of viral infectious diseases and / or autoimmune diseases of novel coronavirus, influenza virus, RSV virus, and the proportion of fucose deficiency is calculated by normalizing the sum of the signal intensities of fucose-deficient glycoforms G1, G1N, G2, G2S to the intensity of the IgG1-GPSVFPLAPSSK peptide segment, and the proportion of galactose deficiency is calculated by normalizing the sum of the signal intensities of galactose-deficient glycoforms G0F, G0NF to the intensity of the IgG1-GPSVFPLAPSSK peptide segment.

6. The MRM quantitative analysis method for the glycosylation of the Fc fragment of the specific antibody according to claim 1, characterized in that In step (5), the liquid chromatography conditions include: an ultra-high performance liquid chromatography system; a reverse-phase chromatographic column; a water / acetonitrile mobile phase; gradient elution.

7. The method for MRM quantitative analysis of the glycosylation of the Fc segment of the specific antibody according to claim 6, wherein In step (5), the liquid chromatography conditions include: an Acquity PREMIER Peptide BEH C18 chromatographic column; column temperature: 40 °C; the mobile phase includes phase A: 0.1% formic acid-aqueous solution and phase B: 0.1% formic acid-acetonitrile solution; flow rate: 0.3 mL / min; gradient elution is carried out according to the following conditions: the gradient starts from 2% of mobile phase B, holds for 0.5 min, then at 9.0 min, the percentage of phase B increases to 38%, then at 10.5 min, it increases to 95%, holds until 12.5 min; finally at 12.6 min, the percentage of phase B drops to 2% and holds for 2.4 min, and the total running time is 15 min.

8. The MRM quantitative analysis method for the glycosylation of the Fc segment of the specific antibody according to claim 1, characterized in that In step (5), the triple quadrupole mass spectrometry conditions include: an ESI ion source, positive ion mode, and MRM detection mode.

9. Use of the method for quantitatively analyzing the glycosylation of the Fc segment of the specific antibody according to any one of claims 1 to 8 in the analysis of serum specific antibody subtypes and glycoforms in patients with viral infectious diseases and / or autoimmune diseases including novel coronavirus, influenza virus, and RSV virus.

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