A method for enriching trace glycosylated peptides based on lectin-coated magnetic beads

Abstract

The present invention provides a method for enriching trace glycosylated peptides based on lectin-coated magnetic beads. First, the lectin is coated on the surface of the magnetic beads through a non-covalent binding method, and then the lectin-coated magnetic beads are used to specifically enrich glycosylated peptides. The present invention mainly improves the method for enriching glycosylated peptides based on lectin. The entire experimental process is carried out in a reaction tube, with simple operation, which helps to reduce sample loss. This method is particularly suitable for enriching N-glycosylated peptides in trace samples. Combined with a high-resolution mass spectrometer, it can realize the automated large-scale identification of N-glycosylated peptides in trace samples based on the characteristics of magnetic beads. Therefore, the present invention provides a method for enriching N-glycosylated peptides with strong generality and high sensitivity, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of glycosylated proteomics, and particularly relates to a method for enriching trace glycosylated peptides based on lectin-coated magnetic beads. Background Art

[0002] Glycosylation is one of the most common and abundant post-translational modifications of proteins, and more than half of the proteins in mammalian cells are glycosylated. In addition, glycoproteins have important clinical diagnostic value, and most of the protein disease markers currently used for disease diagnosis are glycoproteins. N-glycosylation is an important type of glycosylation. N-glycosylation modification occurs on the specific amino acid sequence N-X!-S / T of proteins (where X represents any amino acid other than proline), and the glycan is linked to the protein through the NH2 of the asparagine residue. N-glycosylation is very important for protein folding, protein stability, and protein transport, and also participates in various biological processes such as cell signal transduction, cell adhesion, and the interaction between sperm and oocytes. The occurrence and development of various diseases such as diabetes, cancer, Alzheimer's disease, autoimmune diseases, and inflammation are regulated by N-glycoproteins or accompanied by abnormal N-glycosylation. Therefore, in-depth study of N-glycosylation is of great significance for understanding protein function and the pathogenesis of diseases. However, among the peptides after whole protease digestion, the proportion of glycopeptides is only 2% - 5%. Due to the interference of a large number of non-glycosylated modified peptides, it is very difficult to directly send the sample for mass spectrometry detection and identify glycoproteins. Therefore, it is necessary to enrich glycopeptides first before mass spectrometry detection to achieve the separation of glycopeptides and non-glycopeptides, reduce the interference of non-glycosylated peptides on the mass spectrometry detection process, and thus improve the sensitivity of mass spectrometry detection.

[0003] Since glycosylated modified proteins account for a relatively low proportion in total proteins, glycopeptide enrichment is a key step in glycosylated proteomics research. Currently, common N-glycopeptide enrichment methods include lectin affinity chromatography (LAC), hydrophilic interaction chromatography (HILIC), hydrazide chemical enrichment method, and borate-based enrichment method. However, these methods usually require hundreds of micrograms of protein or peptides as the starting amount, which is not suitable for the research of rare samples such as oocytes. Therefore, it is an urgent need to develop a method for glycosylated proteomics research applicable to trace samples. Herein, we have developed an N-glycosylated proteomics method based on lectin-coated magnetic beads applicable to trace samples. Summary of the Invention

[0004] The object of the present invention is to fill the defect of low sensitivity in the existing N-glycosylated peptide enrichment methods, and to provide a method for enriching trace glycosylated peptides based on lectin-coated magnetic beads; this method develops a new method for enriching N-glycosylated peptides that is applicable to trace samples, has simple steps, is convenient to operate, has high specificity, and has universality, and can realize the enrichment and identification of glycosylated peptides in trace samples.

[0005] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0006] The present invention protects a method for enriching trace glycosylated peptides based on lectin-coated magnetic beads. The method uses magnetic beads as the matrix material, and realizes the highly sensitive enrichment of glycosylated peptides through enrichment operations in a single tube and the high affinity between lectin and glycopeptides.

[0007] In a specific embodiment, the method includes the following steps: (1) In an organic solution at neutral pH, magnetic beads capture lectin and coat it on the surface of the magnetic beads in a non-covalent binding manner; (2) Wash the lectin that has not bound to the magnetic beads; (3) Incubate the peptide sample with the magnetic beads coated with lectin to specifically enrich glycosylated peptides; (4) After washing the non-glycopeptides that have not bound to the magnetic beads, add N-glycanase F (PNGase F) to release the N-glycosylated peptides from the magnetic beads. During this process, the asparagine at the glycosylation modification site will be converted to aspartic acid, resulting in a mass shift (+0.984 Da); (5) Elute the peptides under acidic conditions and perform mass spectrometry detection; (6) When retrieving the mass spectrometry data, add a variable modification of deamidation (asparagine is converted to aspartic acid, Asn->Asp, molecular weight +0.984 Da) to obtain N-glycosylated proteomics data.

[0008] In a specific embodiment, the ratio of magnetic beads to lectin in step (1) is between 10:1 and 1:2 (m / m), specifically 10:1, 5:1, 2:1, 1:1, 1:2, etc.

[0009] In a specific embodiment, the reagent used in step (1) is one of organic solvents with the same properties such as ethanol and acetonitrile, and the concentration range of the organic solvent is ≥50%, such as 50% ethanol solution, 60% ethanol solution, 80% ethanol solution, 50% acetonitrile solution, 60% acetonitrile solution, 80% acetonitrile solution, etc.

[0010] In a specific embodiment, the lectin in step (1) is one or a mixture of several lectins such as concanavalin A (ConA), wheat germ agglutinin (WGA), and ricin agglutinin (RCA).

[0011] In a specific embodiment, the magnetic beads in step (1) can be carboxyl or amino modified magnetic beads.

[0012] In a specific embodiment, the reagent used in step (2) is one of the same property organic solvents such as ethanol and acetonitrile, and the concentration range of the organic solvent is ≥50%, such as 50% ethanol solution, 60% ethanol solution, 80% ethanol solution, 50% acetonitrile solution, 60% acetonitrile solution, 80% acetonitrile solution, etc.

[0013] In a specific embodiment, the ratio of the lectin-coated magnetic beads to the peptide segment in step (3) is between 20:1 and 1:1, specifically such as 20:1, 10:1, 5:1, 2:1, 1:1, etc.

[0014] In a specific embodiment, the amount of the peptide segment sample in step (3) is between 10 μg and 200 μg.

[0015] In a specific embodiment, the incubation conditions in step (3) are an organic solvent greater than 50%, the temperature is room temperature, and the time is 3 - 12 hours.

[0016] In a specific embodiment, the non-glycopeptide washing reagent used in step (4) is one of the same property organic solvents such as ethanol and acetonitrile, and the concentration range of the organic solvent is ≥50%, such as 50% ethanol solution, 60% ethanol solution, 80% ethanol solution, 50% acetonitrile solution, 60% acetonitrile solution, 80% acetonitrile solution, etc.

[0017] In a specific embodiment, the PNGase F treatment time in step (4) is 3 - 12 hours, and the temperature is 25 - 37°C.

[0018] In a specific embodiment, the elution condition in step (5) is acidic (pH < 3).

[0019] Compared with the prior art, the present invention has the following remarkable advantages: (1) It is a universal method that can specifically enrich N-glycosylated peptide segments in different types of samples. (2) It is applicable to the enrichment of N-glycosylated peptide segments in trace samples (such as 10 μg peptide segments). (3) The whole process is carried out in one tube, avoiding problems such as serious sample loss caused by sample transfer and the use of different materials, and realizing the enrichment of glycopeptides in a single centrifuge tube. (4) The lectin is coupled with the magnetic beads in a non-covalent manner, and the activity of the lectin is not affected during this process, and it has a strong enrichment efficiency. (5) The magnetic beads have magnetism, combined with an automated magnetic bead instrument, automated operation can be realized, and the sample preparation efficiency can be improved. Description of the Drawings

[0020] Figure 1 It is a flow chart for enriching glycosylated peptide segments.

[0021] Figure 2 SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining analysis results of lectins captured by magnetic beads and lectins not captured by magnetic beads under the action of organic solvents.

[0022] Figure 3 Numbers of identified N-glycoproteins and N-glycosylation sites at different ratios of magnetic beads to lectins and different ratios of peptide segments to lectins.

[0023] Figure 4 Venn diagram of the numbers of N-glycoproteins and N-glycosylation sites identified from three biological replicates using 10 μg of mouse testis peptide segments.

[0024] Figure 5 Numbers of identified N-glycoproteins and N-glycosylation sites without enrichment of glycosylated peptide segments and with enrichment of N-glycosylated peptide segments using N-Glyco-FASP method, hydrazide chemical enrichment method, and lectin-coated magnetic beads, respectively.

[0025] Figure 6 Venn diagram of the numbers of N-glycoproteins and N-glycosylation sites identified from three biological replicates using 1000 mouse oocytes. Detailed implementation manners

[0026] The technical solutions of the present invention will be further described below with reference to the accompanying drawings. Reagents or instruments and equipment not indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0027] As Figure 1 shown, first, in an ethanol solution at neutral pH, carboxyl-modified magnetic beads capture lectin and immobilize it on the surface of the magnetic beads, and the lectin not bound to the magnetic beads is washed. Then, the peptide segment sample is incubated with the carboxyl-modified magnetic beads bound with lectin for enrichment of glycosylated peptide segments. After washing the non-glycopeptides not bound to the magnetic beads, glycosidase PNGase F is added to release the N-glycosylated peptide segments from the magnetic beads. During this process, asparagine at the glycosylation modification site is converted to aspartic acid, resulting in a mass shift (+0.984 Da). Finally, the peptide segments are eluted under acidic conditions for mass spectrometry detection.

[0028] The following examples illustrate the preferred specific implementation manners of the present invention, but the present invention is not limited thereto.

[0029] Example 1 Capture of lectin by carboxyl-modified magnetic beads

[0030] 1 Preparation of lectin solution. Weigh 5 mg of ConA using an analytical balance and add it to 1 mL of the pre-prepared lectin storage solution. (Lectin storage solution formula: 10 mM NaHCO3, 150 mM NaCl, 0.1 mM CaCl2, 0.01 mM MnCl2, pH = 8.0).

[0031] 2 Washing of beads. Pipette 40 μg of carboxyl-modified magnetic beads into a clean 1.5 mL low-protein adsorption centrifuge tube, add 100 μL of ultrapure water, gently shake and mix well, adsorb the magnetic beads with a magnetic stand, and discard the liquid.

[0032] 3 Add 20 μg of lectin ConA to a 1.5 mL low-protein adsorption centrifuge tube according to the ratio of magnetic beads:lectin = 2:1 (m / m); add an appropriate amount of ultrapure water to make up to 50 μL, add 50 μL of absolute ethanol, gently shake and mix well. Incubate with a shaking mixer at 1000 rpm at room temperature for 20 min under 50% ethanol conditions.

[0033] 4 After incubation, adsorb the magnetic beads with a magnetic stand, discard the supernatant, add 200 μL of 80% ethanol to wash the beads, adsorb the magnetic beads with a magnetic stand, discard the liquid, and repeat 3 times. Thus, magnetic beads with lectin linked to carboxyl-modified magnetic beads are prepared.

[0034] 5 Results: As Figure 2 shown, under the induction of the organic solvent ethanol, after sufficient reaction, almost all ConA binds to the carboxyl-modified magnetic beads, and very little ConA remains in the supernatant.

[0035] Example 2 Use the magnetic beads with lectin linked to carboxyl-modified magnetic beads prepared in Example 1 as a glycopeptide enrichment material for the enrichment and optimization of mouse testicular glycosylated peptide segments

[0036] 1 According to the ratio of lectin to peptide sample (m / m) of 1:1, 2:1, 5:1, 10:1, 20:1, add lectin according to the amount of the peptide sample for testing. Adjust the ratio of carboxyl-modified magnetic beads to lectin (m / m) to 1:2, 1:1, 2:1, 5:1, 10:1 for testing.

[0037] 2 Sequentially add 10 μg of mouse testicular peptide sample, 2 μL of 1 M CaCl2, 2 μL of 1 M MnCl2, 2 μL of 1 M MgCl2 to the carboxyl-modified magnetic beads bound with lectin, add 140 μL of absolute ethanol, and finally make up to 200 μL with ultrapure water. Incubate on a rotary shaker at 10 rpm at room temperature for 3 h.

[0038] 3 After incubation, adsorb the magnetic beads with a magnetic stand and discard the supernatant. Then add 0.5 mL of 80% ethanol solution, invert the magnetic beads up and down to mix well, adsorb the magnetic beads with a magnetic stand, discard the liquid, and repeat 6 times.

[0039] Add 100 U of PNGase F, 50 μL of 20 mM Tris·HCl (pH = 7.5), mix well using a shaker at 1000 rpm, incubate at 37 °C for 3 h. After incubation, add 10 μL of 5% FA, adsorb the magnetic beads using a magnetic stand, retain the liquid, and perform desalting.

[0040] Add 10 μL of 5% FA to each tube, adsorb the magnetic beads using a magnetic stand, and perform desalting using StageTips (Thermo, USA).

[0041] 6 Results: As Figure 3 shown, when the ratio of lectin ConA to the peptide sample is 2:1 (m / m), and the ratio of carboxyl-modified magnetic beads to lectin is 2:1 (m / m), the number of N-glycoproteins and N-glycosylation sites identified is significantly higher than that of other groups, with a maximum of 540 N-glycoproteins and 974 N-glycosylation sites identified.

[0042] Example 3 Use the magnetic beads obtained by linking the lectin prepared in Example 1 with carboxyl-modified magnetic beads as the glycopeptide enrichment material and the optimized conditions in Example 2 for the enrichment of glycosylated peptides in mouse testis.

[0043] 1 Add 10 μg of mouse testis peptide sample, 2 μL of 1 M CaCl2, 2 μL of 1 M MnCl2, 2 μL of 1 M MgCl2, and 140 μL of absolute ethanol to the carboxyl-modified magnetic beads conjugated with lectin in sequence, and finally make up to 200 μL with ultrapure water. Incubate on a rotary shaker at 10 rpm at room temperature for 3 h.

[0044] 2 After incubation, adsorb the magnetic beads using a magnetic stand and discard the supernatant. Then add 0.5 mL of 80% ethanol solution, mix the magnetic beads by inverting up and down, adsorb the magnetic beads using a magnetic stand, discard the liquid, and repeat 6 times.

[0045] 3 Add 100 U of PNGase F, 50 μL of 20 mM Tris·HCl (pH = 7.5), mix well using a shaker at 1000 rpm, incubate at 37 °C for 3 h. After incubation, add 10 μL of 5% FA, adsorb the magnetic beads using a magnetic stand, retain the liquid, and perform desalting.

[0046] 4 Add 10 μL of 5% FA to each tube, adsorb the magnetic beads using a magnetic stand, and perform desalting using StageTips.

[0047] 5 Results: As Figure 4As shown, from 10 μg of mouse testicular peptides, with three biological replicates, a total of 708 N-glycoproteins and 1420 N-glycosylation sites were identified. The results indicate that the lectin-coated magnetic bead-based microscale glycopeptide enrichment method can enrich N-glycopeptides from a peptide sample as low as 10 μg.

[0048] As Figure 5 shown, without glycopeptide enrichment, only about 30 N-glycoproteins and 40 N-glycosylation sites can be identified. The lectin-coated magnetic bead-based microscale glycopeptide enrichment method significantly improves the identification numbers of N-glycoproteins and N-glycosylation sites in the sample.

[0049] As Figure 5 shown, compared with the traditional N-Glyco-FASP and hydrazide chemical enrichment methods, the lectin-coated magnetic bead-based microscale glycopeptide enrichment method significantly improves the identification numbers of both N-glycoproteins and N-glycosylation sites.

[0050] Example 4: The magnetic beads with lectin linked to carboxyl-modified magnetic beads prepared in Example 1 were used as glycopeptide enrichment materials for the enrichment of glycopeptides from mouse oocytes.

[0051] 1. To the carboxyl-modified magnetic beads conjugated with lectin, successively add the peptide sample prepared from 1000 mouse oocytes, 2 μL of 1 M CaCl2, 2 μL of 1 M MnCl2, 2 μL of 1 M MgCl2, and 140 μL of absolute ethanol, and finally make up to 200 μL with ultrapure water. Incubate on a rotary shaker at 10 rpm at room temperature for 3 h.

[0052] 2. After incubation, adsorb the magnetic beads with a magnetic stand and discard the supernatant. Then add 0.5 mL of 80% ethanol solution, mix the magnetic beads by inverting up and down, adsorb the magnetic beads with a magnetic stand, discard the liquid, and repeat 6 times.

[0053] 3. Add 100 U of PNGase F and 50 μL of 20 mM Tris·HCl (pH = 7.5), mix with a vortex mixer at 1000 rpm, incubate at 37 °C for 3 h. After incubation, add 10 μL of 5% FA, adsorb the magnetic beads with a magnetic stand, retain the liquid, and perform desalting.

[0054] 4. Add 10 μL of 5% FA to each tube, adsorb the magnetic beads with a magnetic stand, and desalt using a StageTip.

[0055] 5. Results: As Figure 6As shown, a method for enriching trace glycosylated peptides based on lectin-coated magnetic beads identified a total of 215 N-glycoproteins and 363 N-glycosylation sites from 1000 mouse oocytes with three biological replicates. The results showed that the method for enriching trace glycosylated peptides based on lectin-coated magnetic beads could enrich N-glycosylated peptides from as few as 1000 mouse oocyte samples.

[0056] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, all changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are taken as the protection scope.

Claims

1. A method for enriching trace glycosylated peptide segments based on lectin-coated magnetic beads, which is used for glycoproteomics analysis, and is characterized in that, The method includes the following steps: (1) In an organic solvent at neutral pH, magnetic beads capture lectin and coat it on the surface of the magnetic beads in a non-covalent binding manner; (2) Wash the lectin that has not bound to the magnetic beads; (3) Incubate the peptide sample with the magnetic beads coated with lectin to enrich the glycosylated peptides; (4) After washing the non-glycopeptides that have not bound to the magnetic beads, add N-glycosidase F to release the N-glycosylated peptides from the magnetic beads. During this process, the asparagine at the glycosylation modification site will be converted to aspartic acid, resulting in a mass shift; (5) Elute the peptides under acidic conditions and perform mass spectrometry detection; (6) When retrieving the mass spectrometry data, add the variable modification of deamidation to obtain the N-glycoproteomics data; Among them, the organic solvent used in steps (1)-(4) is ethanol or acetonitrile, and the concentration of the organic solvent is 50-80%; The lectin in step (1) is concanavalin A.

2. A method for enriching trace glycosylated peptide segments based on lectin-coated magnetic beads according to claim 1, wherein In step (1), the mass ratio of the magnetic beads to the lectin is between 10:1 and 1:

2.

3. A method for enriching trace glycosylated peptide segments based on lectin-coated magnetic beads according to claim 1, characterized in that, The magnetic beads in step (1) are carboxyl- or amino-modified magnetic beads.

4. A method for enriching trace glycosylated peptide segments based on lectin-coated magnetic beads according to claim 1, characterized in that In step (3), the mass ratio of the magnetic beads coated with lectin to the peptides is between 20:1 and 1:

1.

5. A method for enriching trace glycosylated peptide segments based on lectin-coated magnetic beads according to claim 1, characterized in that, The amount of the peptide sample in step (3) is between 10 μg and 200 μg.

6. The method for enriching trace glycosylated peptide segments based on lectin-coated magnetic beads according to claim 1, wherein The incubation conditions in step (3): the temperature is room temperature and the time is 3-12 hours.

7. A method for enriching trace glycosylated peptide segments based on lectin-coated magnetic beads according to claim 1, characterized in that In step (4), the treatment time of N-glycosidase F is 3-12 hours and the temperature is 25-37°C.

8. A method for enriching trace glycosylated peptide segments based on lectin-coated magnetic beads according to claim 1, characterized in that, The elution conditions in step (5) are acidic, pH < 3.

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