Method for identifying glycosylation modification of immunoglobulin M

Abstract

The invention discloses a method for identifying glycosylation modification of immunoglobulin M, and belongs to the technical field of protein analysis. The method comprises the following steps: (1) separating IgM from a sample; (2) preparation and enrichment of IgM glycopeptide; and (3) detecting the IgM glycopeptide by mass spectrometry. Two-dimensional electrophoretic separation is carried out on blood / plasma and other samples containing IgM by combining native-PAGE gel electrophoresis and SDS-PAGE gel electrophoresis, the separation method is mild, the glycosylation rate of the obtained IgM is high, and a good foundation is laid for high-coverage identification of subsequent glycosylation modification; by optimizing the enrichment conditions and elution conditions of the modified polydopamine magnetic nanomaterial, high-coverage enrichment of glycopeptides at five N-glycosylation sites of IgM is realized, and the method has great significance in early warning of immune inflammatory diseases, disease progress monitoring, prognosis evaluation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of protein analysis, and more particularly to a method for identifying glycosylation modification of immunoglobulin M. Background Art

[0002] Immunoglobulin (Ig) refers to a globulin with antibody activity or a chemical structure similar to that of an antibody molecule. It is a tetrapeptide chain composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds. Immunoglobulins are divided into five categories: immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE). Immunoglobulin M (IgM) is the largest immunoglobulin in the human circulatory system. It is primarily synthesized by plasma cells in the spleen and lymph nodes and distributed as a pentamer in the blood, cerebrospinal fluid, and lymph, accounting for 5% to 10% of the total Ig in the blood. IgM is produced in the early stages of the adaptive immune response and has powerful functions in pathogen clearance, complement activation, immune regulation, and agglutination. It is also involved in pathological processes such as autoimmune diseases, hypersensitivity reactions, and immune tolerance.

[0003] IgM has complex glycosylation modifications, and its heavy chain contains five independent N-glycosylation sites, which are modified by complex, hybrid, and highly mannose N-glucans. Among them, Asn-171, Asn-332, and Asn-395 sites are mainly modified by complex N-glucans, while Asn-402 and Asn-563 sites are mainly modified by high-mannose N-glucans. These site-specific glycosylation modifications have an important impact on the folding, conformation, distribution, stability, and biological functions of IgM: the inhibition of N-glycan synthesis in IgM causes B cells to almost not secrete IgM; increased IgM sialylation leads to enhanced T cell inhibition; IgM N-glycans interact with C1q and mannan-binding lectin (MBL) related to the complement activation lectin pathway in the classical complement pathway; sialylation and mannosylation modifications of IgM are associated with the severity of COVID-19 infection and the complement deposition rate.

[0004] However, due to the high molecular weight, low abundance, low stability and high heterogeneity of its glycosylation modification, IgM faces multiple challenges in the development of separation and purification and glycosylation modification identification technologies: (1) In terms of separation and purification, gel filtration technology has insufficient resolution and is prone to forming polymers or binding to other proteins; although affinity chromatography technology can achieve efficient enrichment of IgM, it is expensive and the antibody specificity needs to be strictly verified; (2) In terms of glycopeptide enrichment, the lectin method is expensive and has poor versatility; the hydrazine chemical method has relatively harsh reaction conditions and sugar chains are easily lost during the enrichment process; the boric acid method can form a boronate structure with cis-vicinal diols to capture glycopeptides, but it is time-consuming; (3) In terms of glycosylation modification identification, IgM has five glycosylation sites, and its N-glycan types include high mannose type, hybrid type and complex type. Currently, the glycosylation analysis of IgM in clinical samples has problems such as low sensitivity, low throughput and low glycoform coverage.

[0005] Therefore, developing a method to separate IgM from complex biological samples such as plasma / serum and enrich and identify its site-specific glycosylation modifications is important for analyzing the structure-activity relationship of IgM glycosylation modifications, exploring the mechanism of disease occurrence and development, disease prognosis, diagnosis, and drug treatment response judgment. Summary of the Invention

[0006] To address the above problems, the present invention provides a method for identifying glycosylation modifications of IgM.

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

[0008] A method for identifying glycosylation modification of IgM, comprising the following steps:

[0009] (1) Isolation of IgM from samples

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

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

[0012] The gel band containing the IgM 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 with a modified polydopamine magnetic nanomaterial to obtain an IgM glycopeptide;

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

[0014] IgM 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: 5 μL of IgM sample is added to 5 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 IgM 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 5% isocratic native-PAGE gel, and electrophoresis is performed at a constant voltage of 220 V under ice bath conditions for 60 min.

[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 5% isocratic native-PAGE gel is composed of 5% separating gel (water: 30% acrylamide solution: Tris-borate-magnesium chloride buffer = 5.6 / 1.5 / 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).

[0018] Native-PAGE can achieve the separation of native conformation proteins under non-denaturing conditions without the use of denaturants such as SDS or reducing agents such as DTT. However, the natural charge, molecular size and shape can affect the mobility of proteins in native-PAGE. Therefore, using IgM standard as a marker molecule, or isocratic (5%) native-PAGE electrophoresis is used to obtain the band containing pentameric IgM in the IgM-containing sample, while removing high-abundance blood proteins with similar molecular weights such as albumin (~70kDa) and transferrin (~80kDa), which greatly reduces the complexity of subsequent SDS-PAGE separation samples, avoids interference from transferrin, etc., and improves the IgM separation resolution. Each gel can achieve the separation of up to 14 samples.

[0019] Preferably, the SDS-PAGE gel electrophoresis in step (1) is as follows: the native-PAGE gel strip containing IgM 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.

[0020] Preferably, the reduction and alkylation treatment is as follows: after native-PAGE electrophoresis of 5 μL of IgM sample, the native-PAGE gel strip containing IgM is cut out, 3 ml of 0.2 M 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 0.5 M IAA solution is added for alkylation reaction at room temperature in the dark for 1 hour.

[0021] 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.

[0022] Preferably, the 5% SDS-PAGE stacking gel consists 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.

[0023] Preferably, the in-gel enzymatic hydrolysis in step (2) is as follows: grinding the IgM heavy chain SDS-PAGE gel band corresponding to the IgM sample into pieces of about 0.1 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 1 hour, add 25 mM ammonium bicarbonate solution until the gel surface is covered, react at 37 ° C for 16 hours, add deionized water to mix, centrifuge at low speed, and take the supernatant.

[0024] Preferably, in the in-gel enzymatic digestion, based on 5 μL of IgM sample corresponding to the IgM heavy chain SDS-PAGE gel band, 25 ng / μL trypsin solution (prepared with 25 mM ammonium bicarbonate solution): 25 mM ammonium bicarbonate solution: deionized water = 10 μL: 40 μL: 150 μL.

[0025] In-gel enzymatic hydrolysis is carried out in a neutral and weakly alkaline environment, avoiding the need to replace the acidic solution environment with a weakly alkaline environment during the immunoadsorption method for IgM separation, and minimizing the loss of terminal sialic acid.

[0026] Preferably, the specific operation of reducing and alkylating the gel strip in step (1) is as follows: cutting the gel strip containing IgM and placing it in a 12-well plate, adding 3 mL of 0.2 M DTT solution and performing a reduction reaction at 37° C. for 1 hour, discarding the DTT solution and washing the gel strip three times with ultrapure water; then adding 3 mL of 0.5 M IAA solution and performing an alkylation reaction at room temperature in the dark for 1 hour.

[0027] Preferably, the enrichment material in step (2) is a modified polydopamine magnetic nanomaterial, which has been disclosed in the patent application document CN117736480A (application publication date: March 22, 2024). The material is obtained by polyurea modification of polydopamine-coated magnetic spheres at room temperature to obtain modified polydopamine magnetic spheres with stronger hydrophilicity, negative surface charge, and increased specific surface area, thereby achieving high selectivity, high sensitivity, low cost, and no destruction of the integrity of the glycan structure of intact glycopeptides such as IgG and fetuin. The specific preparation method of the modified polydopamine magnetic nanomaterial is as follows:

[0028] 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 with N,N-dimethylformamide three times to obtain polyurea-modified polydopamine magnetic beads; disperse in water for more than 3 hours to obtain modified polydopamine magnetic nanomaterials.

[0029] Preferably, the enrichment in step (2) comprises the following steps: (a) washing the modified polydopamine magnetic nanomaterial three times with an enrichment solution and resuspending the solution to prepare an enrichment suspension with a final concentration of 1 mg / mL, mixing the solution with the dried IgM enzymatic hydrolysate in proportion, vortexing to dissolve the solution, oscillating to enrich the solution for 1 hour, magnetically separating the solution and discarding the enrichment suspension, washing the solution with 85% acetonitrile three times, magnetically separating the solution again and discarding the washing solution;

[0030] (b) The magnetic nanomaterial washed in step (a) is dispersed in an eluent in proportion, shaken and eluted for 45 minutes, and the eluted liquid is collected to obtain the IgM glycopeptide.

[0031] Preferably, the composition of the enrichment solution in step (a) is: 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, v / v / v); the ratio of the dried IgM enzymatic hydrolysate to the enrichment suspension is 5 μg:100 μL; and the oscillation enrichment conditions are 25°C and 1000 rpm.

[0032] Preferably, the composition of the eluent in step (b) is: 10% acetonitrile + 0.0125% ammonia water (acetonitrile / water / ammonia water = 10 / 89.9875 / 0.0125); the amount of the eluent added is: based on 5 μg of dry IgM enzymatic hydrolysate, add 100 μL of eluent; the conditions for the oscillation elution are 25°C and 1000 rpm.

[0033] The present invention achieves high coverage enrichment of glycopeptides at the five N-glycosylation sites of IgM by optimizing the enrichment conditions and elution conditions of modified polydopamine magnetic nanomaterials.

[0034] 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.

[0035] Preferably, the sample loaded for mass spectrometry analysis is obtained by the following treatment: the IgM glycopeptide obtained in step (2) is centrifuged and concentrated at 25°C and 1500 rpm for 90 min, 5 μL of deionized purified water is added at a solid-liquid ratio of 1:1 to dissolve it, 0.5 μL is spotted on the MTP AnchorChip™ target, 0.5 μL of 10 mg / mL CHCA solution is added, mixed thoroughly, and evaporated naturally.

[0036] High-resolution Fourier transform ion cyclotron resonance mass spectrometer can ensure efficient qualitative and quantitative analysis of intact glycopeptides due to its advantages such as high detection mass accuracy, high throughput, high vacuum, and single charge.

[0037] Preferably, in any of the above methods for identifying glycosylation modifications of immunoglobulin M, the IgM-containing sample comprises any one of serum, plasma, cerebrospinal fluid, and joint fluid.

[0038] Proteins in 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.

[0039] Beneficial effects of the present invention:

[0040] Compared with commercially available IgM affinity purification kits (such as POROS TM CaptureSelect TMCompared with IgM-XL affinity matrix, ThermoScientific, price 9660 RMB / 5mL, product number 2812892005), the IgM separation method provided by the present invention has mild separation conditions (immunoaffinity method requires elution with citric acid solution at pH 3.5, which easily leads to inactivation or loss of glycopeptides);

[0041] The molecular weight of IgM obtained by the immunoaffinity method in the prior art (“Comprehensive Immunoglobulin G, A, and MGlycopeptide Profiling for Large-Scale Biomedical Research” van Tol BDM, Wasynczuk AM, Gijze S, et al, Mol Cell Proteomics. 2025, 24(3), 100928) was about 70 kDa after SDS detection. However, as can be seen from Example 1, the molecular weight of IgM obtained by the present invention was about 80 kDa after SDS detection, indicating that the glycosylation rate of IgM obtained by the separation method of the present invention is high, which lays a good foundation for the subsequent high-coverage identification of glycosylation modifications.

[0042] Compared to commercially available HILIC SPE glycopeptide enrichment materials, the glycopeptide enrichment material provided by the present invention is inexpensive, readily available, simple to operate, and offers high enrichment sensitivity and identification coverage. By optimizing the enrichment and elution conditions of the modified polydopamine magnetic nanomaterial, the present invention achieves high-coverage enrichment of glycopeptides at the five N-glycosylation sites of IgM, which has significant implications for early warning of immune-inflammatory diseases, monitoring disease progression, and prognostic assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : Comparison of gel images obtained after native-PAGE electrophoresis separation of plasma IgM and human IgM standards;

[0044] Figure 2 : Comparison of gel images obtained after SDS-PAGE electrophoresis separation of native-PAGE gel bands containing IgM in plasma and human IgM standard;

[0045] Figure 3 : Glycopeptide mass spectra of IgM enzymatic hydrolysate after enrichment with enrichment buffers of different acidities, wherein: (a) 85% acetonitrile: enrichment buffer with a composition of 85% acetonitrile (acetonitrile / water = 85 / 15, v / v); (b): enrichment buffer with a composition of 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, v / v / v); (c): enrichment buffer with a composition of 85% acetonitrile + 0.2% formic acid (acetonitrile / water / formic acid = 85 / 14.8 / 0.2, v / v / v);

[0046] Figure 4 : Glycopeptide mass spectra of IgM enzymatic hydrolysate after elution with different eluents, (a) H2O: eluent consisting of H2O; (b) 0.0125% ammonia: eluent consisting of 0.0125% ammonia; (c) 10% acetonitrile + 0.0125% ammonia: eluent consisting of 10% acetonitrile + 0.0125% ammonia; (d): eluent consisting of 25% acetonitrile + 0.0125% ammonia;

[0047] Figure 5 : Comparison of the corresponding mass spectrometry detection of IgM enzymatic hydrolysis products after enrichment and without enrichment, wherein a is the direct mass spectrometry detection of IgM enzymatic hydrolysis products; b is the representative mass spectrum of IgM glycopeptides obtained after enrichment of IgM enzymatic hydrolysis products;

[0048] Figure 6 : Comparison of mass spectrometry detection effects of IgM enzymatic hydrolysis products 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;

[0049] Figure 7 : Mass spectra of IgM hydrolysis products with different contents after enrichment by modified polydopamine magnetic nanomaterials (Fe3O4@PDA@DETA), including: (a) the hydrolysis product prepared by 500fmol: 5μg IgM (SDS-PAGE gel band of IgM heavy chain), (b) the hydrolysis product prepared by 250fmol: 2.5μg IgM (SDS-PAGE gel band of IgM heavy chain), (c) the hydrolysis product prepared by 50fmol: 0.5μg IgM (SDS-PAGE gel band of IgM heavy chain), and (d) the hydrolysis product prepared by 10fmol: 0.1μg IgM (SDS-PAGE gel band of IgM heavy chain). DETAILED DESCRIPTION

[0050] 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.

[0051] 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.

[0052] The following reagents are illustrative:

[0053] Plasma samples (Cayman Chemical, Humanplasma, cat. no. 400100-500).

[0054] Pancreatin solution (Promega, cat. no. V5113)

[0055] 2× native-PAGE loading buffer: A trace amount of xylene cyanol FF was dissolved in a buffer (0.2 M Tris-HCl (pH 7.5): glycerol: water = 1 / 2 / 7, v / v / v).

[0056] 5% isocratic native-PAGE gel: composed of 5% separating gel (water: 30% acrylamide solution: Tris-borate-magnesium chloride buffer = 5.6 / 1.5 / 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).

[0057] Contains 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.

[0058] 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.

[0059] Protein electrophoresis PAGE gel rapid staining solution (catalog number 4306120-500) was purchased from Beijing Jude Antai Technology Co., Ltd.

[0060] Example 1 Isolation of IgM from a Sample Containing IgM

[0061] Using plasma as the sample containing IgM, the following experiment was performed:

[0062] 1. Native-PAGE gel electrophoresis experiment

[0063] Human IgM standard was used as a molecular marker, and native-PAGE gel electrophoresis was performed on the plasma sample. The specific operation was as follows: 5 μL of plasma sample was added to 5 μL of 2×native-PAGE loading buffer at a volume ratio of 1:1 and mixed thoroughly as the experimental sample. At the same time, 10 μL of 1 mg / mL human IgM standard (Sigma I8260 IgM) solution was mixed thoroughly with 10 μL of 2×native-PAGE loading buffer as the molecular marker sample. The samples were loaded onto a 5% isocratic native-PAGE gel, and electrophoresis was performed at a constant voltage of 220V for 60 minutes under ice bath conditions. After the electrophoresis, the sample was stained for 30 minutes (using protein electrophoresis PAGE gel rapid staining solution), and then repeatedly decolorized with ultrapure water until the gel background was colorless. The migration pattern of IgM in native-PAGE was observed by the native-PAGE gel chromatograms of plasma samples and human IgM standards. Figure 1 The gel chromatograms of three repeated native-PAGE experiments of plasma IgM and human IgM standards show that native-PAGE can retain the pentameric structure of IgM intact at the top of the separation gel under ice bath conditions. Subsequent analysis of this band can greatly reduce the complexity of subsequent protein separation.

[0064] 2. SDS-PAGE gel electrophoresis experiment

[0065] Cut the above-mentioned native-PAGE gel strip containing IgM and place it in a 12-well plate. Add 3mL of 0.2M DTT solution and reduce it at 37℃ for 1h. Discard the DTT solution and wash the strip three times with ultrapure water. Then add 3mL of 0.5M IAA solution and alkylate it at room temperature in the dark for 1h. Place the treated native-PAGE strip on a glass plate containing 8% SDS-PAGE separation gel, with the lower edge of the strip about 1cm away from the upper edge of the separation gel. Then pour 5% SDS-PAGE stacking gel. After the stacking gel solidifies, run electrophoresis at 80V for 30min and then at 160V for 60min. After the electrophoresis, stain it for 30min (use protein electrophoresis PAGE gel rapid staining solution). Then decolorize it repeatedly with ultrapure water until the gel background is colorless. Figure 2 The gel chromatograms of three replicates of SDS-PAGE of plasma IgM and human IgM standards are shown. As can be seen, an IgM heavy chain band was detected at a molecular weight of approximately 80 kDa by comparison with the human IgM standard.

[0066] In summary, the separation method of the present invention has good stability, and the purity of the separated IgM heavy chain is sufficient to meet the requirements of subsequent glycosylation modification identification.

[0067] Example 2 Preparation and Enrichment of IgM Glycopeptides

[0068] 1. Preparation of IgM Glycopeptides

[0069] (1) The SDS-PAGE band obtained in Example 1 corresponding to the molecular weight of 80 kDa was cut to obtain the SDS-PAGE band of IgM heavy chain, and the band was cut into pieces of about 0.1 mm using a grinder. 3 After the micelles were prepared, 200 μL of 50% acetonitrile decolorizing solution (prepared with 25 mM ammonium bicarbonate solution) was added and the mixture was shaken and decolorized for 30 minutes at 25°C and 1000 rpm on a homogenizer. The decolorizing solution was discarded and the decolorization was repeated twice. The micelles were dehydrated with acetonitrile and then freeze-dried in a vacuum at 25°C and 1500 rpm for 5 minutes to completely dehydrate the micelles. 10 μL of 25 ng / μL pancreatic enzyme solution (prepared with 25 mM ammonium bicarbonate solution) was added dropwise. After incubation at 4°C for 90 minutes, 40 μL of 25 mM ammonium bicarbonate solution was added and the enzymatic hydrolysis reaction was carried out at 37°C for 16 hours. 150 μL of deionized water was added and mixed thoroughly. 100 μL of the supernatant was collected and concentrated by vacuum centrifugation at 25°C and 1500 rpm for 90 minutes to obtain the IgM dry enzymatic hydrolysate for later use.

[0070] 2. Enrichment of IgM glycopeptides

[0071] (1) Preparation of enrichment materials

[0072] 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, which are used for the enrichment of IgM glycopeptides.

[0073] (2) Optimization of enrichment conditions

[0074] ① Optimization of enrichment solution

[0075] 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 IgM enzymatic hydrolysate. The specific procedures are as follows:

[0076] 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.

[0077] 5 μg of IgM dried enzymatic hydrolysate was taken respectively, and 100 μL of the above-mentioned different enrichment suspensions were added to each. After vortexing to dissolve, the IgM enzymatic hydrolysate solution was obtained, and the solution was placed on a mixer and shaken at 25°C and 1000 rpm for 1 hour. The enrichment suspension was magnetically separated and discarded. 200 μL of 85% acetonitrile was used as a washing liquid to wash the magnetic nanomaterials after magnetic separation. The mixture was vortexed for 20 seconds, magnetically separated again, and the washing liquid was discarded. The above washing operation was repeated twice. The washed magnetic nanomaterials were dispersed in 100 μL of 10% acetonitrile + 0.0125% ammonia water (acetonitrile / water / ammonia water = 10 / 89.9875 / 0.0125) and placed on a homogenizer for 45 min of oscillation at 25°C and 1000 rpm for elution. 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 min before being used as a glycopeptide sample for mass spectrometry detection.

[0078] 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.

[0079] Test results are shown in Figure 3 ,Depend on Figure 3It can be seen that compared with the other two enrichment solutions, the enrichment solution using 85% acetonitrile + 0.1% formic acid enriched more glycopeptides and had stronger signals, indicating that the enrichment suspension prepared with 85% acetonitrile + 0.1% formic acid as the enrichment solution and modified polydopamine magnetic nanomaterials has the best effect on enriching IgM glycopeptides.

[0080] ②Optimization of eluent

[0081] Based on 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, v / v / v) as the enrichment solution, H2O, 0.0125% ammonia water, 10% acetonitrile + 0.0125% ammonia water (acetonitrile / water / ammonia water = 10 / 89.9875 / 0.0125, v / v / v) and 25% acetonitrile + 0.0125% ammonia water (acetonitrile / water / ammonia water = 25 / 74.9875 / 0.0125, v / v / v) were used as eluents to enrich the IgM enzymatic hydrolysate. The specific operation is as follows:

[0082] The modified polydopamine magnetic nanomaterials were washed three times with 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, v / v / v) and resuspended to prepare an enrichment suspension with a final concentration of 1 mg / mL for standby use. 5 μg of IgM dry enzymatic hydrolysate was taken, 100 μL of the above enrichment suspension was added to each, and the IgM enzymatic hydrolysate solution was obtained after vortexing and dissolving. The solution was placed on a homogenizer and shaken at 25°C and 1000 rpm for 1 hour. The solution was magnetically separated and the enrichment suspension was discarded. 200 μL of 85% acetonitrile was used as a washing solution to wash the magnetic nanomaterials after magnetic separation. The mixture was vortexed for 20 seconds, magnetically separated again and the washing solution was discarded. The above washing operation was repeated twice. 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.

[0083] 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 compared with the other three eluents, when 10% acetonitrile + 0.0125% ammonia water was used as the eluent, the signal intensity of glycopeptides with a mass-to-charge ratio above 4000 was the best, which was conducive to the high coverage identification of IgM glycosylation modification, indicating that the use of 10% acetonitrile + 0.0125% ammonia water as the eluent had the best enrichment effect on IgM glycopeptides.

[0084] In summary, the best enrichment effect of IgM glycopeptide was achieved by using 85% acetonitrile + 0.1% formic acid as the enrichment solution and the enrichment suspension prepared with modified polydopamine magnetic nanomaterials, and 10% acetonitrile + 0.0125% ammonia water as the eluent.

[0085] (3) Comparison of mass spectrometry results before and after IgM glycopeptide enrichment

[0086] The modified polydopamine magnetic nanomaterials were washed three times with 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, 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 the dried IgM hydrolysate, vortexed to dissolve, and the resulting IgM hydrolysate solution was placed on a homogenizer and shaken at 25°C, 1000 rpm for 1 hour. The enrichment suspension was magnetically separated and discarded. The magnetic nanomaterials after magnetic separation were washed with 200 μL of 85% acetonitrile as a washing solution, vortexed for 20 seconds, magnetically separated again, and the washing solution discarded. The above washing operation was repeated twice. The washed magnetic nanomaterials were dispersed in 100 μL of 10% acetonitrile + 0.0125% ammonia water and placed on a mixer for 45 min of oscillation at 25°C and 1000 rpm for elution. 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 min, and then used as the experimental group glycopeptide sample for mass spectrometry detection.

[0087] Another 5 μg of IgM dry enzymatic hydrolysate was added with 5 μL of deionized pure water as the blank control group glycopeptide sample for mass spectrometry detection.

[0088] 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 (SolariXFTICRMS, 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 5 ,Depend on Figure 5It can be seen that in the mass spectrum of direct detection of IgM enzymatic hydrolysis products (5a), only a small amount of glycopeptide signals marked with "★" can be seen in the high molecular weight (2500-3500Da) region; in the mass spectrum of the IgM enzymatic hydrolysis products obtained after enrichment (5b), there is basically no non-glycopeptide signal in the mass spectrometry detection signal, and 102 IgM glycopeptides corresponding to five glycosylation sites can be detected in the high molecular weight region (2500-8000Da), including 20 at the Asn171[N46] site, 39 at the Asn332[N209] site, 35 at the Asn395[N272]-Asn402[N279] site, and 8 at the Asn563[N439] site. This shows that the method of the present invention can detect IgM glycopeptides with high coverage.

[0090] Example 3 Verification of the enrichment effect of modified polydopamine magnetic nanomaterials on IgM glycopeptides

[0091] 1. Comparison of enrichment effect with commercially available HILICSPE columns

[0092] Glycopeptide enrichment was performed on the dried IgM enzymatic hydrolysate (prepared as described in Example 2, 1. IgM glycopeptide) 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 two activation cycles, 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 IgM enzymatic hydrolysate (prepared as described in Example 2, 1.IgM glycopeptide) was dissolved in 200 μL of an 86% acetonitrile aqueous solution containing 1% trifluoroacetic acid to obtain an enzymatic hydrolysate solution. The sample was loaded three times, and the column was washed by adding 1 mL of an 86% acetonitrile aqueous solution containing 1% trifluoroacetic acid. After washing three times, 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 detection.

[0093] 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 FTICRMS, 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. 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.

[0094] Test results are shown in Figure 5 By comparing the two glycopeptide enrichment methods, it can be found that the mass spectrum obtained by enrichment with the materials and methods of the present invention has a clean background, a wide variety of IgM glycopeptides, and a strong signal. The mass spectrum obtained by enrichment with a commercially available HILIC SPE column only shows a small number of glycopeptide peaks with low signal intensity, indicating that the enrichment method of the present invention has a good enrichment effect on IgM glycopeptides and a high identification coverage rate.

[0095] 2. Detection of the Capture Capacity of Modified Polydopamine Magnetic Nanomaterials for IgM Glycopeptides

[0096] The modified polydopamine magnetic nanomaterials were washed three times with 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, 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 each of the dried enzymatic hydrolysates obtained from different IgM masses (IgM heavy chain gel bands obtained by SDS-PAGE) and vortexed to dissolve the resulting IgM hydrolysate solutions.

[0097] The solutions of the above-mentioned IgM 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 magnetically separated and discarded. The residue was washed three times with 200 μL of 85% acetonitrile, vortexed for 20 seconds, and magnetically separated again, and the wash solution was discarded. This was repeated three times. 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 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.

[0098] 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.

[0099] Test results are shown in Figure 7 ,Depend on Figure 7 It can be seen that glycopeptide information appears when the molar mass of the enzymatic hydrolysis product solution is 10 fmol, and the glycopeptide information is rich and the glycopeptide signal reaches a high intensity at 500 fmol. The detection limit of IgM glycopeptide can be as low as 10 fmol, indicating that the modified polydopamine magnetic nanomaterial has a high capture ability for IgM glycopeptide under the optimized enrichment conditions, which can ensure the detection sensitivity of glycosylation modification of IgM in plasma samples.

[0100] 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.

[0101] 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 identifying glycosylation modification of immunoglobulin M, characterized in that: The following steps are involved: (1) Isolation of IgM from IgM-containing samples Using human IgM standard as a molecular marker, the IgM-containing samples were subjected to native-PAGE gel electrophoresis and SDS-PAGE gel electrophoresis, and the gel band containing the IgM heavy chain was cut out; (2) Preparation and enrichment of IgM glycopeptides The gel band containing the IgM 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 with a modified polydopamine magnetic nanomaterial to obtain an IgM glycopeptide; (3) Mass spectrometry detection of IgM glycopeptides IgM glycopeptides were detected using high-resolution Fourier transform ion cyclotron resonance mass spectrometry.

2. The method for identifying glycosylation modification of immunoglobulin M according to claim 1, characterized in that: The native-PAGE gel electrophoresis in step (1) is as follows: 5 μL of IgM sample is added to 5 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 IgM 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 5% isocratic native-PAGE gel, and electrophoresis is performed at a constant voltage of 220 V in an ice bath for 60 min.

3. The method for identifying glycosylation modification of immunoglobulin M according to claim 1, characterized in that: The SDS-PAGE gel electrophoresis in step (1) is as follows: the native-PAGE gel strip containing IgM that has been reduced and alkylated is placed on a glass plate containing 8% SDS-PAGE separation gel, and a 5% SDS-PAGE stacking gel is poured. After the stacking gel solidifies, electrophoresis is performed at 80V for 30 minutes, and then at 160V for 60 minutes.

4. The method for identifying glycosylation modification of immunoglobulin M according to claim 1, characterized in that: The in-gel enzymatic digestion in step (2) is as follows: the IgM heavy chain SDS-PAGE band corresponding to the IgM sample is ground into pieces of about 0.1 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 solution 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 identifying glycosylation modification of immunoglobulin M according to claim 4, characterized in that: In the in-gel digestion, based on the IgM heavy chain SDS-PAGE gel band corresponding to 5 μL of IgM sample, 25 ng / μL trypsin solution (prepared with 25 mM ammonium bicarbonate solution): 25 mM ammonium bicarbonate solution: deionized water = 10 μL: 40 μL: 150 μL.

6. The method for identifying glycosylation modification of immunoglobulin M 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. The mixture was then mixed with the dried IgM enzymatic hydrolysate in a certain proportion, vortexed to dissolve, and concentrated by oscillation for 1 hour. The enrichment suspension was then magnetically separated and discarded. The mixture was then washed three times with 85% acetonitrile, magnetically separated again, and the washing solution was discarded. (b) The magnetic nanomaterial washed in step (a) is dispersed in an eluent in proportion, shaken and eluted for 45 minutes, and the eluted liquid is collected to obtain the IgM glycopeptide.

7. The method for identifying glycosylation modification of immunoglobulin M according to claim 6, characterized in that: The composition of the enrichment solution in step (a) is: 85% acetonitrile + 0.1% formic acid (acetonitrile / water / formic acid = 85 / 14.9 / 0.1, v / v / v); The ratio of the dried IgM enzymatic hydrolysate to the enriched suspension is 5 μg:100 μL; The conditions for the oscillation enrichment were 25° C. and 1000 rpm.

8. The method for identifying glycosylation modification of immunoglobulin M according to claim 6, characterized in that: The composition of the eluent in step (b) is: 10% acetonitrile + 0.0125% ammonia water (acetonitrile / water / ammonia water = 10 / 89.9875 / 0.0125, v / v / v); The amount of the eluent added is: based on 5 μg of dry IgM enzymatic hydrolysate, add 100 μL of eluent; The shaking elution conditions were 25° C. and 1000 rpm.

9. The method for identifying glycosylation modification of immunoglobulin M 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 identifying glycosylation modification of immunoglobulin M 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.

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