Method for high-throughput synchronous analysis of complete O-glycopeptide and N-glycopeptide of immunoglobulin A

Abstract

The invention discloses a method for high-throughput synchronous analysis of complete O-glycopeptide and N-glycopeptide of immunoglobulin A, and belongs to the technical field of protein analysis. The method comprises the following steps: (1) separating IgA from a sample; (2) preparation and enrichment of IgA glycopeptide; and (3) detecting the IgA glycopeptide by mass spectrometry. According to the method, IgA is separated and purified through electrophoresis, IgA glycopeptides are enriched through a nano material, accurate molecular mass information (observed values) and information provided by a database are obtained through a high-resolution mass spectrum, corresponding possible glycopeptides are speculated, the theoretical molecular weight of the glycopeptides is calculated, and 93 kinds of glycopeptides including N-glycopeptides and O-glycopeptides are totally identified. According to the method, complete N glycopeptides and O glycopeptides of IgA in serum / plasma and other IgA-containing body fluid samples can be simultaneously determined, and information of glycoforms and glycosylation sites on the glycopeptides is reserved. The method has the advantages of strong operability, high sensitivity, good accuracy and good stability.

Description

Technical Field

[0001] The present invention relates to the technical field of protein analysis, and more particularly to a method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A. Background Art

[0002] Immunoglobulin A (IgA) is a soluble glycoprotein widely distributed on mucosal surfaces, including those in the respiratory and digestive tracts, and in body fluids. Its glycosylation pattern (including N-glycosylation and O-glycosylation) directly regulates inflammatory responses and pathogen clearance. N-glycosylation of IgA occurs at asparagine (Asn) residues in the CH2 domain of the heavy chain constant region. By adding complex glycan chains, it maintains IgA stability and its interaction with Fc receptors (such as FcαRI). Sialylation of N-glycans regulates IgA binding to the hepatocyte sialoglycoprotein receptor (ASGPR), affecting its half-life and clearance pathway. Studies have shown that desialylated IgA is taken up by hepatocytes and excreted into the intestine via bile, participating in the mucosal immune system. Furthermore, abnormal N-glycosylation may impair IgA binding to immune cells, leading to immune escape or autoimmune diseases such as IgA nephropathy.

[0003] O-glycosylation of IgA occurs primarily on serine / threonine residues in the hinge region of the IgA1 isoform, forming short glycan chains (such as GalNAc and galactose). This modification enhances the hinge region's resistance to protease degradation and maintains IgA1 stability in the mucosal environment. Abnormal O-glycosylation (such as galactose deficiency) also plays a core role in the development of IgA nephropathy (IgAN). Defective O-glycans expose antigenic epitopes, inducing the formation of autoantibodies (such as anti-glycosylated IgA antibodies), which form immune complexes that deposit in the glomeruli, triggering inflammation and renal damage. Furthermore, O-glycans participate in the binding of IgA to pathogens (such as bacterial proteins). By hijacking IgA's O-glycans as attachment sites, they affect its neutralizing capacity and weaken its immune protective function. Therefore, the precise regulation of O-glycosylation has dual implications for IgA's mucosal defense and pathological processes.

[0004] Currently, analytical techniques for IgA glycopeptide levels mainly include glycopeptide enrichment technology and mass spectrometry identification technologies such as LC-MS and MALDI-TOF based on liquid chromatography (LC) separation. However, they still face the problem of difficulty in simultaneous analysis of N-glycopeptides and O-glycopeptides. Since N-glycosylation (asparagine linkage) and O-glycosylation (serine / threonine linkage) have significant differences in biological functions, and the differences in their structural complexity, dynamic range and abundance pose challenges to analysis. In terms of enrichment strategies, lectin affinity chromatography can selectively enrich specific glycoforms, but its specificity for complex samples is insufficient and it cannot distinguish between N / O-glycosylation; hydrophilic interaction chromatography (HILIC) utilizes the difference in hydrophilicity of glycopeptides for enrichment, but has low sensitivity for low-abundance O-glycopeptides. In terms of mass spectrometry analysis, due to the differences in mass spectrometry sensitivity of different instruments, N-glycopeptides are easy to detect due to their larger sugar chains, while O-glycopeptides have short sugar chains and low abundance and are easily masked by background signals. In short, N-glycosylation sites are relatively conservative and the enrichment methods are mature, while O-glycosylation lacks universal enzymatic tools and enrichment strategies, resulting in independent analysis processes for the two, requiring multiple experiments, low efficiency and high sample consumption.

[0005] Therefore, establishing an efficient co-analysis method for intact N / O-glycopeptides of immunoglobulin A is of great significance to promoting the application of glycoproteomics in clinical diagnosis and treatment. Summary of the Invention

[0006] To address the above problems, the present invention provides a method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A, comprising:

[0009] (1) Isolation of IgA from IgA-containing samples

[0010] Using human IgA standard as a molecular marker, the IgA-containing samples were subjected to native-PAGE gel electrophoresis and SDS-PAGE gel electrophoresis, and the gel band containing the IgA heavy chain was cut out;

[0011] (2) Preparation and enrichment of IgA glycopeptides

[0012] The gel band containing the IgA heavy chain obtained in step (1) is subjected to in-gel enzymatic digestion using pancreatic enzyme, and then concentrated by vacuum centrifugation to obtain a dry enzymatic hydrolysis product, which is then enriched using a modified polydopamine magnetic nanomaterial to obtain an IgA glycopeptide;

[0013] (3) Mass spectrometry detection of IgA glycopeptides

[0014] IgA glycopeptides were detected using high-resolution Fourier transform ion cyclotron resonance mass spectrometry.

[0015] Preferably, the native-PAGE gel electrophoresis in step (1) is as follows: 2 μL of IgA sample is added to 8 μL of 2x native-PAGE loading buffer and mixed thoroughly as an experimental sample; at the same time, 10 μL of 1 mg / mL human IgA standard solution is taken and mixed thoroughly with 10 μL of 2x native-PAGE loading buffer as a molecular marker sample, and the samples are loaded onto a 7.5% isocratic native-PAGE gel, and electrophoresis is performed at a constant voltage of 100 V for 15 minutes under ice bath conditions, and then the voltage is adjusted to 200 V for constant voltage electrophoresis for 45 minutes.

[0016] Preferably, the 2× native-PAGE loading buffer is composed of a trace amount of xylene cyanol FF dissolved in a buffer (0.2 M Tris-HCl (pH 7.5): glycerol: water = 1 / 2 / 7 (v / v / v).

[0017] Preferably, the 7.5% isocratic native-PAGE gel consists of 7.5% separating gel (water: 30% acrylamide solution: Tris-borate-magnesium chloride buffer = 4.85 / 2.25 / 1.8, v / v / v) and 4% stacking gel (water: 30% acrylamide solution: 0.2 M Tris-HCl (pH 7.5) = 15 / 2.7 / 2, v / v / v).

[0018] Preferably, the SDS-PAGE gel electrophoresis is as follows: the alkylated native-PAGE gel strip containing IgA is placed on a glass plate containing 8% SDS-PAGE separation gel, 5% SDS-PAGE stacking gel is poured, and after the stacking gel solidifies, electrophoresis is performed at 80V for 30 minutes and then at 160V for 60 minutes.

[0019] Preferably, the 8% SDS-PAGE separation gel consists of water: 30% acrylamide solution: 1.5M Tris-HCl (pH 8.8): 10% SDS = 4.6 / 2.7 / 2.5 / 0.1, v / v / v / v.

[0020] Preferably, the 5% SDS-PAGE stacking gel is composed of water: 30% acrylamide solution: 1.5M Tris-HCl (pH=6.8): 10% SDS=6.8 / 1.7 / 1.25 / 0.1, v / v / v.

[0021] Preferably, the reduction and alkylation treatment is as follows: after native-PAGE electrophoresis of 2 μL of IgA-containing sample, the native-PAGE gel strip containing IgA is cut, 3 ml of 0.03 g / mL DTT solution is added for reduction reaction at 37° C. for 1 hour, the DTT solution is discarded and the gel strip is washed three times with ultrapure water; then 3 mL of 0.12 g / mL IAA solution is added for alkylation reaction at 37° C. in the dark for 2 hours.

[0022] Preferably, the in-gel enzymatic hydrolysis in step (2) is as follows: grinding the IgA heavy chain SDS-PAGE band corresponding to the IgA sample into pieces of about 0.2 mm 3 After decolorization and complete dehydration, add 25 ng / μL trypsin solution (prepared with 25 mM ammonium bicarbonate solution) to the gel particles, incubate at 4°C for 90 min, add 25 mM ammonium bicarbonate until the gel surface is covered, react at 37°C for 16 h, add deionized water to mix, centrifuge at low speed, and take the supernatant.

[0023] Preferably, in the in-gel enzymatic hydrolysis, based on the IgA heavy chain SDS-PAGE band corresponding to 2 μL of IgA sample, the addition ratio of each reagent is 20 μL of trypsin solution (prepared with 25 mM ammonium bicarbonate solution): 40 μL of 25 mM ammonium bicarbonate solution: 150 μL of deionized water.

[0024] Preferably, the centrifugation in step (2) is: centrifugal concentration at 25° C. and 1500 rpm for 90 min.

[0025] Preferably, the enrichment in step (2) comprises the following steps:

[0026] (a) The modified polydopamine magnetic nanomaterial was washed three times with an enrichment solution and resuspended to prepare an enrichment suspension with a final concentration of 1 mg / mL, which was then mixed with the dried IgA enzymatic hydrolysate in a certain proportion, vortexed to dissolve, and then vibrated for enrichment for 1 hour. The enrichment suspension was then magnetically separated and discarded, and the enrichment suspension was washed three times with 85% acetonitrile, magnetically separated again, and the washing solution was discarded;

[0027] (b) The magnetic nanomaterials washed in step (a) were dispersed in an eluent in proportion, shaken and eluted for 45 minutes, and the eluted liquid was collected.

[0028] Preferably, the composition of the enrichment solution in step (a) is: 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v); the ratio of the dried IgA enzymatic hydrolysate to the enrichment suspension is 5 μg:100 μL; and the oscillation enrichment is at 25°C and 1000 rpm.

[0029] Preferably, the eluent in step (b) is a 25% acetonitrile aqueous solution; the amount of the eluent added is: based on 5 μg of dry IgA enzymatic hydrolysate, add 100 μL of eluent; the conditions for the oscillation elution are 25° C. and 1000 rpm.

[0030] Preferably, the mass spectrometry detection conditions in step (3) are: positive ion mode, laser power 50%, acquisition range 1000-8000Da; mass selection window Q value 2700-3500, time of flight TOF 3.0-3.5ms; data information in the mass spectrum is selected based on signal-to-noise ratio ≥3, relative intensity >0.1%, absolute intensity >100000, and reliable isotope peaks.

[0031] Preferably, in any of the above-mentioned methods for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A, the IgM-containing sample comprises any one of serum, plasma, cerebrospinal fluid, and joint fluid.

[0032] Proteins in body fluids such as serum, plasma, cerebrospinal fluid, and synovial fluid have certain similarities in molecular weight and charge distribution, so similar separation effects can be achieved using the same Native-PAGE conditions.

[0033] The present invention has the following beneficial effects: The analytical method provided herein separates and purifies IgA through electrophoresis, enriches IgA glycopeptides using nanomaterials, and utilizes high-resolution mass spectrometry to obtain accurate molecular mass information (observed values) and database information to infer corresponding glycopeptides and calculate their theoretical molecular weights. A total of 93 glycopeptides, including N- and O-glycopeptides, were identified. This method can simultaneously measure intact IgA N- and O-glycopeptides in IgA-containing body fluids such as serum and plasma, while retaining information on the glycoforms and glycosylation sites on each glycopeptide. The method is highly operable, sensitive, accurate, and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Comparison of gel images obtained after native-PAGE electrophoresis separation of plasma IgA and human IgA standards;

[0035] Figure 2 : Comparison of gel images obtained after SDS-PAGE electrophoresis separation of native-PAGE gel bands containing IgA in plasma and human IgA standard;

[0036] Figure 3: Glycopeptide mass spectra of IgA hydrolysate after enrichment with enrichment buffers of different acidities, wherein: (a) 85% acetonitrile: enrichment buffer is 85% acetonitrile (acetonitrile / water = 85 / 15, v / v); (b) 85% acetonitrile + 0.1% formic acid: enrichment buffer is 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, v / v / v); (c) 85% acetonitrile + 0.2% formic acid: enrichment buffer is 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v);

[0037] Figure 4 : Glycopeptide mass spectra of IgA enzymatic hydrolysate after enrichment and elution with different eluents, (a): H2O: eluent is H2O, (b) 25% acetonitrile: eluent is 25% acetonitrile aqueous solution, (c): 50% acetonitrile: eluent is 50% acetonitrile aqueous solution;

[0038] Figure 5 : Comparison of the corresponding mass spectrometry detection of IgA hydrolysis products after enrichment and without enrichment, wherein a is the direct mass spectrometry detection diagram of IgA hydrolysis products; b is the representative mass spectrum of IgA glycopeptides obtained after enrichment of IgA hydrolysis products with modified polydopamine magnetic nanomaterials;

[0039] Figure 6 : Comparison of mass spectrometry detection effects of IgA hydrolysate after enrichment with different glycopeptide enrichment materials, (a) Fe3O4@PDA@DETA: enrichment was performed using modified polydopamine magnetic nanomaterials (Fe3O4@PDA@DETA), (b) HILIC SPE: enrichment was performed using commercially available HILIC SPE glycopeptide enrichment materials;

[0040] Figure 7 : Mass spectra of IgA hydrolysis products with different contents after enrichment by modified polydopamine magnetic nanomaterials (Fe3O4@PDA@DETA), including: (a) 5 μg of the IgA heavy chain gel band obtained by SDS-PAGE of 3000 fmol:10 μg human IgA standard, (b) 1 μg of the IgA heavy chain gel band obtained by SDS-PAGE of 600 fmol:10 μg human IgA standard, (c) 0.2 μg of the IgA heavy chain gel band obtained by SDS-PAGE of 120 fmol:10 μg human IgA standard, and (d) 0.1 μg of the IgA heavy chain gel band obtained by SDS-PAGE of 60 fmol:10 μg human IgA standard. DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0042] The reagents involved in the embodiments of the present invention were all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0043] The following reagents are illustrative:

[0044] Plasma samples (Cayman Chemical, Humanplasma, cat. no. 400100-500).

[0045] Trypsin solution (Promega, cat. no. V5113).

[0046] 2× native-PAGE loading buffer: A trace amount of xylene cyanol FF dissolved in a buffer (0.2 M Tris-HCl (pH 7.5): glycerol: water = 1 / 2 / 7 (v / v / v).

[0047] 7.5% isocratic native-PAGE gel: composed of 7.5% separating gel (water: 30% acrylamide solution: Tris-borate-magnesium chloride buffer = 4.85 / 2.25 / 1.8, v / v / v) and 4% stacking gel (water: 30% acrylamide solution: 0.2M Tris-HCl (pH 7.5) = 15 / 2.7 / 2, v / v / v).

[0048] 8% SDS-PAGE separation gel: composed of water: 30% acrylamide solution: 1.5 M Tris-HCl (pH 8.8): 10% SDS = 4.6 / 2.7 / 2.5 / 0.1 (v / v / v / v).

[0049] 5% SDS-PAGE stacking gel: composed of water: 30% acrylamide solution: 1.5 M Tris-HCl (pH = 6.8): 10% SDS = 6.8 / 1.7 / 1.25 / 0.1 (v / v / v).

[0050] Protein electrophoresis PAGE gel rapid staining solution (catalog number 4306120-500) was purchased from Beijing Jude Antai Technology Co., Ltd.

[0051] Example 1 Isolation of IgA from a Sample Containing IgA

[0052] Using plasma as the sample containing IgA, the following experiment was performed:

[0053] 1. Native-PAGE gel electrophoresis experiment

[0054] Human IgA standard was used as a molecular marker, and native-PAGE gel electrophoresis was performed on the plasma sample. The specific operation was as follows: 2 μL of plasma sample was added to 8 μL of 2×native-PAGE loading buffer and mixed thoroughly as the experimental sample. At the same time, 10 μL of 1 mg / mL human IgA standard (Sigma I4036 IgA) solution was taken and mixed thoroughly with 10 μL of 2×native-PAGE loading buffer as the molecular marker sample. The samples were loaded onto 7.5% isocratic native-PAGE gel, and electrophoresis was performed at a constant voltage of 100 V for 15 minutes under ice bath conditions. The voltage was adjusted to 200 V constant voltage electrophoresis for 45 minutes. After the electrophoresis was completed, staining was performed for 30 minutes (using protein electrophoresis PAGE gel rapid staining solution), and then decolorization was repeated with ultrapure water until the gel background was colorless. The migration pattern of IgA in native-PAGE was observed through the native-PAGE gel chromatograms of plasma samples and human IgA standards. Figure 1 As shown in the gel chromatograms of three repeated native-PAGE experiments of plasma IgA and human IgA standards, native-PAGE separates IgA from transferrin and proteins under ice-bath conditions while retaining the IgA structure intact in the separation gel. Subsequent analysis of the native-PAGE bands corresponding to IgA can greatly reduce the complexity of subsequent protein separation.

[0055] 2. SDS-PAGE gel electrophoresis experiment

[0056] Cut the above-mentioned native-PAGE gel strip containing IgA and place it in a 12-well plate. Add 3 ml of 0.03 g / mL DTT solution and carry out reduction reaction at 37°C for 1 hour. Discard the DTT solution and wash the strip three times with ultrapure water. Then add 3 mL of 0.12 g / mL IAA solution and carry out alkylation reaction at 37°C in the dark for 2 hours. Place the treated native-PAGE strip on a glass plate containing 8% SDS-PAGE separation gel, with the lower edge of the strip about 1 cm away from the upper edge of the separation gel. Then pour 5% SDS-PAGE stacking gel. After the stacking gel solidifies, perform electrophoresis at 80V for 30 minutes and then at 160V for 60 minutes. After the electrophoresis, stain for 30 minutes (use protein electrophoresis PAGE gel rapid staining solution), and then decolorize repeatedly with ultrapure water until the gel background is colorless. Figure 2The gel chromatograms of three repeated SDS-PAGE experiments of native-PAGE gel bands containing IgA in plasma IgA and human IgA standards are shown. As can be seen from the comparison with the human IgA standard, an IgA heavy chain band was detected at a molecular weight of approximately 70 kDa.

[0057] In summary, the separation method of the present invention has good stability, and the purity of the separated IgA heavy chain is sufficient to meet the requirements of subsequent glycosylation modification identification.

[0058] Example 2 Preparation and Enrichment of IgA Glycopeptides

[0059] 1. Preparation of IgM Glycopeptides

[0060] (1) The SDS-PAGE band obtained in Example 1 corresponding to the molecular weight of 70 kDa was cut to obtain the SDS-PAGE band of IgA heavy chain, and the fragments were ground into pieces of about 0.2 mm 3 The micelles were transferred to a 96-well plate, and 200 μL of 50% acetonitrile decolorizing solution (prepared with 25 mM ammonium bicarbonate solution) was added. The micelles were shaken and decolorized at room temperature for 30 minutes. The decolorizing solution was discarded and the decolorization was repeated twice. The micelles were dehydrated with acetonitrile to make them hard, and then centrifuged and concentrated at 25°C and 1500 rpm for 5 minutes to completely dry the micelles. 20 μL of 25 ng / μL pancreatic enzyme solution (Promega, product number V5113) (prepared with 25 mM ammonium bicarbonate solution) was added and incubated at 4°C for 90 minutes to fully hydrate the micelles. 40 μL of 25 mM ammonium bicarbonate was added until the surface of the micelles was covered. The micelles were reacted at 37°C for about 16 hours. 150 μL of deionized water was added and vortexed to mix well. The mixture was then centrifuged at low speed. 100 μL of the supernatant was taken and concentrated by vacuum centrifugation at 25°C and 1500 rpm for 90 minutes to obtain the IgA dry enzymatic hydrolysate for later use.

[0061] 2. Enrichment of IgA glycopeptides

[0062] (1) Preparation of enrichment materials

[0063] Dissolve 20 mg of dopamine in 10 mL of 10 mM Tris-HCl (pH 8.8), add Fe3O4 magnetic nanomaterials, stir in the dark for 8-24 hours, wash the product and dry it in an oven at 60°C to obtain polydopamine magnetic beads. The washing method is to wash with N,N-dimethylformamide and water alternately three times, and wash with N,N-dimethylformamide twice. Dissolve 16 μL of diethylenetriamine in 2 mL of N,N-dimethylformamide, add 65 mg of N,N-carbonyldiimidazole, and shake at room temperature for 10-60 minutes. Disperse 10 mg of polydopamine nanomaterial magnetic beads in 1 mL of N,N-dimethylformamide, add 8 μL of diethylenetriamine and 1 mL of diethylenetriamine aminoformyl imidazole, and shake at room temperature for 4-20 hours. After magnetic separation, discard the supernatant and wash three times with N,N-dimethylformamide to obtain polyurea-modified polydopamine magnetic beads; disperse in water for more than 3 hours to obtain modified polydopamine magnetic nanomaterials for the enrichment of IgA glycopeptides.

[0064] (2) Optimization of enrichment conditions

[0065] ① Optimization of enrichment solution

[0066] 85% acetonitrile (acetonitrile / water = 85 / 15, v / v), 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, v / v / v), and 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v) were used as enrichment solutions to enrich the IgA enzymatic hydrolysate. The specific operations are as follows:

[0067] The modified polydopamine magnetic nanomaterials were washed three times with the above enrichment solution and resuspended to prepare different enrichment suspensions with a final concentration of 1 mg / mL for later use.

[0068] 5 μg of dried IgA hydrolysate (prepared as described in Example 2 for 1.IgM glycopeptide) were placed in a 96-well plate. 100 μL of each of the above enrichment suspensions was added to each well. The resulting IgA hydrolysate solution was vortexed and placed on a homogenizer at 25°C and 1000 rpm for 1 hour. The enrichment suspension was then magnetically separated and discarded. The magnetic nanomaterials were washed with 200 μL of 85% acetonitrile as a wash solution, vortexed for 20 seconds, magnetically separated again, and the wash solution discarded. This washing process was repeated twice. The washed magnetic nanomaterials were dispersed in 100 μL of 25% acetonitrile aqueous solution and eluted on a homogenizer at 25°C and 1000 rpm for 45 minutes. The magnetic nanomaterials were magnetically separated and discarded. The eluted liquid was collected and concentrated by centrifugation at 25°C and 1500 rpm for 90 minutes before use as a glycopeptide sample for mass spectrometry analysis.

[0069] Take the glycopeptide samples prepared above, add 5 μL of deionized pure water to dissolve each, take 0.5 μL of each and spot on the MTP AnchorChipTM target, mix thoroughly with 0.5 μL of CHCA solution with a concentration of 10 mg / mL, evaporate naturally and perform mass spectrometry analysis. Use a high-resolution Fourier transform ion cyclotron resonance mass spectrometer (SolariX FTICR MS, Bruker, Germany) for detection. The mass spectrometry detection mode is positive ion mode, the laser power is set to 50%, and the acquisition range is set to 1000-8000 Da; the mass selection window Q value is 2700-3500, and the time of flight TOF is 3.0-3.5 ms; the data information (mass-to-charge ratio and relative intensity) in the mass spectrum is obtained using software, and the selection criteria are signal-to-noise ratio ≥3, relative intensity >0.1%, absolute intensity >100000, and reliable isotope peaks; the results are imported into Microsoft Excel for analysis. The test results are shown in Figure 3 ,Depend on Figure 3 It can be seen that compared with the other two enrichment solutions, the glycopeptide signal background enriched by 85% acetonitrile + 0.2% formic acid as the enrichment solution is cleaner, indicating that the enrichment suspension prepared with 85% acetonitrile + 0.2% formic acid as the enrichment solution and modified polydopamine magnetic nanomaterials has the best effect on enriching IgA glycopeptides.

[0070] ②Optimization of eluent

[0071] Based on 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v) as the enrichment solution, H2O, 25% acetonitrile aqueous solution, and 50% acetonitrile aqueous solution were used as eluents to enrich the IgA enzymatic hydrolysate. The specific operation is as follows:

[0072] 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v) modified polydopamine magnetic nanomaterials were washed 3 times and resuspended to prepare an enrichment suspension with a final concentration of 1 mg / mL for standby use. 5 μg of IgA dry hydrolysate (prepared as obtained in Example 2 for 1.IgM glycopeptide) were taken respectively, 100 μL of the above-mentioned enrichment suspension was added to each, vortexed to dissolve to obtain an IgA hydrolysate solution, and placed on a homogenizer and vibrated at 25°C, 1000 rpm for 1 hour, magnetically separated and the enrichment suspension discarded; 200 μL of 85% acetonitrile was used as a washing solution to wash the magnetic nanomaterial after magnetic separation, vortexed for 20 seconds, magnetically separated again and the washing solution discarded, and the above-mentioned washing operation was repeated 2 times. The washed magnetic nanomaterials were dispersed in 100 μL of the above-mentioned different eluents and placed on a mixer for elution at 25°C and 1000 rpm for 45 minutes. The magnetic nanomaterials were magnetically separated and discarded. The eluted liquid was collected and concentrated by centrifugation at 25°C and 1500 rpm for 90 minutes, and then used as the glycopeptide sample for mass spectrometry detection.

[0073] Take the glycopeptide samples prepared above, add 5 μL of deionized pure water to dissolve each, take 0.5 μL of each and spot on the MTP AnchorChipTM target, mix thoroughly with 0.5 μL of CHCA solution with a concentration of 10 mg / mL, evaporate naturally and perform mass spectrometry analysis. Use a high-resolution Fourier transform ion cyclotron resonance mass spectrometer (SolariX FTICR MS, Bruker, Germany) for detection. The mass spectrometry detection mode is positive ion mode, the laser power is set to 50%, and the acquisition range is set to 1000-8000 Da; the mass selection window Q value is 2700-3500, and the time of flight TOF is 3.0-3.5 ms; the data information (mass-to-charge ratio and relative intensity) in the mass spectrum is obtained using software, and the selection criteria are signal-to-noise ratio ≥3, relative intensity >0.1%, absolute intensity >100000, and reliable isotope peaks; the results are imported into Microsoft Excel for analysis. The test results are shown in Figure 4 ,Depend on Figure 4 It can be seen that with the increase of acetonitrile volume fraction, the proportion of glycopeptide signals in the eluted glycopeptides first increases and then decreases. The use of 25% acetonitrile aqueous solution as the eluent has the best enrichment effect on IgA glycopeptides.

[0074] In summary, the best enrichment effect of IgA glycopeptides was achieved by using 85% acetonitrile + 0.2% formic acid as the enrichment liquid and the enrichment suspension prepared with modified polydopamine magnetic nanomaterials, and 25% acetonitrile aqueous solution as the eluent.

[0075] (3) Comparison of mass spectrometry results before and after IgA glycopeptide enrichment

[0076] The modified polydopamine magnetic nanomaterial was washed three times with 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v) and resuspended to prepare an enrichment suspension with a final concentration of 1 mg / mL. 100 μL of the enrichment suspension was added to 5 μg of dried IgA hydrolysate (prepared from 1.1gM glycopeptide in Example 2), vortexed to dissolve, and the resulting IgA hydrolysate solution was placed on a homogenizer and shaken at 25°C, 1000 rpm for 1 hour. The enrichment suspension was then magnetically separated and discarded. 200 μL of 85% acetonitrile was used as a washing solution to wash the magnetic nanomaterial after magnetic separation, vortexed for 20 seconds, magnetically separated again, and the washing solution discarded. The washing operation was repeated twice. The washed magnetic nanomaterials were dispersed in 100 μL of 25% acetonitrile aqueous solution and placed on a mixer for elution at 25°C and 1000 rpm for 45 minutes. The magnetic nanomaterials were magnetically separated and discarded. The eluted liquid was collected and concentrated by centrifugation at 25°C and 1500 rpm for 90 minutes, and then used as the experimental group glycopeptide sample for mass spectrometry detection.

[0077] Another 5 μg of IgA dry enzymatic hydrolysate (obtained from the preparation of 1.IgM glycopeptide in Example 2) was added to 5 μL of deionized purified water to serve as a blank control group glycopeptide sample for mass spectrometry detection.

[0078] Glycopeptide samples from the experimental and blank control groups were dissolved in 5 μL of deionized water. 0.5 μL of each sample was spotted onto an MTP AnchorChip™ target and thoroughly mixed with 0.5 μL of a 10 mg / mL CHCA solution. After evaporation, mass spectrometry analysis was performed using a high-resolution Fourier transform ion cyclotron resonance mass spectrometer (SolariXFTICR MS, Bruker, Germany). The mass spectrometer was operated in positive ion mode, with a laser power of 50% and an acquisition range of 1000–8000 Da. The mass selection window (Q value) was 2700–3500, and the time-of-flight (TOF) was 3.0–3.5 ms. Data (mass-to-charge ratio and relative intensity) from the mass spectra were extracted using software, with selection criteria based on a signal-to-noise ratio ≥ 3, relative intensity > 0.1%, absolute intensity > 100,000, and reliable isotopic peaks. The results were imported into Microsoft Excel for analysis.

[0079] Test results are shown in Figure 5 ,Depend on Figure 5It can be seen that in the mass spectrum of direct detection of IgA hydrolysis products (5a), the signals are mainly non-glycosylated modified peptides in the low molecular weight region (1000-3500 Da), and only a small amount of glycopeptide signals are observed in the high molecular weight region (4000-5500 Da); in the mass spectrum of the IgA hydrolysis products obtained after enrichment (5b), there are basically no non-glycopeptide signals in the mass spectrometry detection signals, and 102 IgA glycopeptides corresponding to five glycosylation sites can be detected in the high molecular weight region, including 41 N-glycosylation sites Asn340 and 52 O-glycosylation sites Ser89-Ser126. This shows that the method of the present invention can simultaneously detect IgA N-glycopeptides and O-glycopeptides in a high-throughput manner.

[0080] Example 3 Verification of the enrichment effect of modified polydopamine magnetic nanomaterials on IgA glycopeptides

[0081] 1. Comparison of enrichment effect with commercially available HILIC SPE columns

[0082] Glycopeptide enrichment was performed on the dried IgA hydrolysate (prepared in 1. Preparation of IgA glycopeptides in Example 2) using a HILIC SPE column (Thermo Fisher Scientific, Cat. No. 60108-364). The specific operation was as follows: 1 mL of 86% acetonitrile aqueous solution containing 1% trifluoroacetic acid (loading buffer) was added to the HILIC SPE column. After the liquid leaked to the adsorption material, the column was gently shaken to activate the HILIC SPE column. After activation was continued twice, 1 mL of water was added to the activated HILIC SPE column. After the liquid leaked to the adsorption material, 1 mL of 86% acetonitrile aqueous solution containing 1% trifluoroacetic acid was added. 5 μg The dried IgA hydrolysate (prepared as described in Example 2 for 1.IgM glycopeptide) was dissolved in 200 μL of an 86% acetonitrile aqueous solution containing 1% trifluoroacetic acid to obtain a hydrolysate solution. The sample was loaded three times, and the column was washed with 1 mL of an 86% acetonitrile aqueous solution containing 1% trifluoroacetic acid. After three washes, 200 μL of water was added for elution. The eluted solution was collected, lyophilized, and used as a positive control glycopeptide sample for mass spectrometry.

[0083] The glycopeptide samples of the positive control group and the experimental group prepared in Example 2 were each dissolved in 5 μL of deionized purified water. 0.5 μL of each sample was then spotted onto an MTP AnchorChip™ target and thoroughly mixed with 0.5 μL of a 10 mg / mL CHCA solution. After evaporation, mass spectrometry analysis was performed using a high-resolution Fourier transform ion cyclotron resonance mass spectrometer (SolariX FTICR MS, Bruker, Germany). The mass spectrometry detection mode was positive ion mode, the laser power was set to 50%, and the acquisition range was set to 1000-8000 Da. The mass selection window Q value was 2700-3500, and the time of flight (TOF) was 3.0-3.5 ms. The data information (mass-to-charge ratio and relative intensity) in the mass spectra was acquired using software, with selection criteria of signal-to-noise ratio ≥3, relative intensity >0.1%, absolute intensity >100,000, and reliable isotopic peaks. The results were imported into Microsoft Excel for analysis.

[0084] Test results are shown in Figure 6 By comparing the two glycopeptide enrichment methods, it can be found that the glycopeptide mass spectrum obtained by enrichment with the materials and methods of the present invention has a clean background, can completely detect the N-glycopeptide and O-glycopeptide of IgA, and has a strong signal. The mass spectrum obtained by enrichment with a commercially available HILIC SPE column only shows a small amount of glycopeptide peaks with low signal intensity, indicating that the enrichment method of the present invention has a good enrichment effect on IgA glycopeptides and comprehensive detection sites.

[0085] 2. Detection of the Capture Capacity of Modified Polydopamine Magnetic Nanomaterials for IgA Glycopeptides

[0086] The modified polydopamine magnetic nanomaterials were washed three times with 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v) and resuspended to prepare an enrichment suspension with a final concentration of 1 mg / mL. 100 μL of the enrichment suspension was added to dried enzymatic hydrolysates prepared from different masses of IgA (IgA heavy chain gel band obtained by SDS-PAGE) at 5 μg, 1 μg, 0.2 μg, and 0.1 μg, respectively. The hydrolysates were vortexed to dissolve the hydrolysates and yield solutions with varying IgA content.

[0087] The solutions of the above-mentioned IgA enzymatic hydrolysates with different contents were placed on a homogenizer and shaken at 25°C and 1000 rpm for 1 hour. The enriched suspension was then magnetically separated and discarded. The magnetic nanomaterials after magnetic separation were washed with 200 μL of 85% acetonitrile as a wash solution, vortexed for 20 seconds, magnetically separated again, and the wash solution discarded. This washing process was repeated twice. The washed magnetic nanomaterials were dispersed in 100 μL of the above-mentioned different eluents and eluted on a homogenizer at 25°C and 1000 rpm for 45 minutes. The magnetic nanomaterials were then magnetically separated and discarded. The eluted liquid was collected and concentrated by centrifugation at 25°C and 1500 rpm for 90 minutes, and then used as the glycopeptide sample for mass spectrometry analysis.

[0088] Each of the glycopeptide samples prepared above was dissolved in 5 μL of deionized water. 0.5 μL of each sample was spotted onto an MTP AnchorChip™ target and thoroughly mixed with 0.5 μL of a 10 mg / mL CHCA solution. After evaporation, mass spectrometry analysis was performed using a high-resolution Fourier transform ion cyclotron resonance mass spectrometer (SolariX FTICR MS, Bruker, Germany). The mass spectrometer was operated in positive ion mode, with a laser power of 50% and an acquisition range of 1000–8000 Da. The mass selection window (Q value) was 2700–3500, and the time-of-flight (TOF) was 3.0–3.5 ms. Data (mass-to-charge ratio and relative intensity) from the mass spectra were extracted using software, with selection criteria based on a signal-to-noise ratio ≥ 3, relative intensity > 0.1%, absolute intensity > 100,000, and reliable isotopic peaks. The results were imported into Microsoft Excel for analysis.

[0089] Test results are shown in Figure 7 ,Depend on Figure 7 It can be seen that when the concentration of the enzymatic hydrolysis product solution is 60 fmol (corresponding to 0.1 μg of the enzymatic hydrolysis product of the IgA heavy chain gel band obtained by SDS-PAGE of 10 μg human IgA standard), glycopeptide information appears. When it is 600 fmol (corresponding to 1 μg of the enzymatic hydrolysis product of the IgA heavy chain gel band obtained by SDS-PAGE of 10 μg human IgA standard), the glycopeptide information is rich and the glycopeptide signal reaches a high intensity. The detection limit of IgA glycopeptide can be as low as 60 fmol, indicating that the modified polydopamine magnetic nanomaterial has a high capture ability for IgA glycopeptides under the optimized enrichment conditions, which can ensure the efficient and synchronous analysis of IgA N-glycopeptides and O-glycopeptides.

[0090] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0091] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A, characterized in that: include: (1) Isolation of IgA from IgA-containing samples Using human IgA standard as a molecular marker, the IgA-containing samples were subjected to native-PAGE gel electrophoresis and SDS-PAGE gel electrophoresis, and the gel band containing the IgA heavy chain was cut out; (2) Preparation and enrichment of IgA glycopeptides The gel band containing the IgA heavy chain obtained in step (1) is subjected to in-gel enzymatic digestion using pancreatic enzyme, and then concentrated by vacuum centrifugation to obtain a dry enzymatic hydrolysis product, which is then enriched using a modified polydopamine magnetic nanomaterial to obtain an IgA glycopeptide; (3) Mass spectrometry detection of IgA glycopeptides IgA glycopeptides were detected using high-resolution Fourier transform ion cyclotron resonance mass spectrometry.

2. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to claim 1, characterized in that: The native-PAGE gel electrophoresis in step (1) is as follows: 2 μL of IgA sample is added to 8 μL of 2x native-PAGE loading buffer and mixed thoroughly as an experimental sample. At the same time, 10 μL of 1 mg / mL human IgA standard solution is mixed thoroughly with 10 μL of 2x native-PAGE loading buffer as a molecular marker sample, and the samples are loaded onto a 7.5% isocratic native-PAGE gel. After electrophoresis at a constant voltage of 100 V for 15 minutes under ice bath conditions, the voltage is adjusted to 200 V and electrophoresis is performed at a constant voltage of 45 minutes.

3. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to claim 1, characterized in that: The SDS-PAGE gel electrophoresis in step (1) is as follows: the native-PAGE gel strip containing IgA that has been reduced and alkylated is placed on a glass plate containing 8% SDS-PAGE separation gel, 5% SDS-PAGE stacking gel is poured, and after the stacking gel solidifies, electrophoresis is performed at 80V for 30 minutes and then at 160V for 60 minutes.

4. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to claim 1, characterized in that: The in-gel enzymatic digestion in step (2) is as follows: the IgA heavy chain SDS-PAGE band corresponding to the IgA sample is ground into pieces of about 0.2 mm 3 After decolorization and complete dehydration, add 25 ng / μL trypsin solution (prepared with 25 mM ammonium bicarbonate solution) to the gel particles, incubate at 4°C for 90 min, add 25 mM ammonium bicarbonate until the gel surface is covered, react at 37°C for 16 h, add deionized water to mix, centrifuge at low speed, and take the supernatant.

5. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to claim 4, characterized in that: In the in-gel enzymatic digestion, based on the IgA heavy chain SDS-PAGE band corresponding to 2 μL of IgA sample, 20 μL of trypsin solution (prepared with 25 mM ammonium bicarbonate solution): 40 μL of 25 mM ammonium bicarbonate solution: 150 μL of deionized water.

6. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to claim 1, characterized in that: The enrichment in step (2) comprises the following steps: (a) The modified polydopamine magnetic nanomaterial was washed three times with an enrichment solution and resuspended to prepare an enrichment suspension with a final concentration of 1 mg / mL, which was then mixed with the dried IgA enzymatic hydrolysate in a certain proportion, vortexed to dissolve, and then vibrated for enrichment for 1 hour. The enrichment suspension was then magnetically separated and discarded, and the enrichment suspension was washed three times with 85% acetonitrile, magnetically separated again, and the washing solution was discarded; (b) The magnetic nanomaterials washed in step (a) are dispersed in an eluent in proportion, shaken and eluted for 45 minutes, and the eluted liquid is collected.

7. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to claim 6, characterized in that: The composition of the enrichment solution in step (a) is: 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v); The ratio of the dried IgA enzymatic hydrolysate to the enriched suspension is 5 μg:100 μL; The shaking enrichment was carried out at 25° C. and 1000 rpm.

8. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to claim 6, characterized in that: The eluent in step (b) is 25% acetonitrile aqueous solution; The amount of the eluent added is: based on 5 μg of dry IgA enzymatic hydrolysate, add 100 μL of eluent; The shaking elution conditions were 25° C. and 1000 rpm.

9. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to claim 1, characterized in that: The mass spectrometry detection conditions described in step (3) are: positive ion mode, laser power 50%, acquisition range 1000-8000Da; mass selection window Q value 2700-3500, time of flight TOF 3.0-3.5ms; data information in the mass spectrum is selected based on signal-to-noise ratio ≥3, relative intensity >0.1%, absolute intensity >100000, and reliable isotope peaks.

10. The method for high-throughput simultaneous analysis of intact O-glycopeptides and N-glycopeptides of immunoglobulin A according to any one of claims 1 to 9, characterized in that: The IgM-containing sample includes any one of serum, plasma, cerebrospinal fluid, and joint fluid.

Images