A method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic cleavage analysis

Abstract

The present invention discloses a method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic digestion analysis, comprising the steps of: extracting proteins, then hydrolyzing the proteins to obtain polypeptides and glycopeptides, and then treating with endoglycosidase, including: ① treating the peptide segments with endoglycosidase, then oxidizing and binding the glycopeptides to hydrazide resin, and obtaining fucosylation sites or defucosylated glycopeptides after hydrolysis by fucosidase; ② treating the glycopeptides with endoglycosidase, and then obtaining intact fucosylated glycopeptides using hydrophilic interaction chromatography. The glycoprotein or enzymatically digested glycopeptides can also be enzymatically digested to obtain branched α1,3-fucosylated glycopeptides after oxidation and binding to hydrazide. Finally, mass spectrometry is used to analyze the fucosylated peptides and the peptides containing fucosylation sites. This method is of great significance for the discovery of fucosylated disease markers in diseased cells, clinical tissues and human body fluid specimens, as well as the research on early disease diagnosis and prognosis markers, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomolecular analysis reagents, and particularly relates to a method for solid-phase fucosylated glycoprotein enrichment and fucosylation cleavage analysis (SPFucE). Background Art

[0002] Glycosylation is an important post-translational modification of proteins and is involved in processes such as growth, differentiation, and inflammation in organisms. Fucose is one of the important carbohydrates in glycan chains. Fucosylation is the process of transferring fucose from GDP-fucose to its substrate under the catalysis of fucosyltransferase. Fucosyltransferase (FUT) is usually involved in the synthesis of terminal glycan structures. Therefore, the transfer of fucose residues to glycans is considered the end point of the glycosylation process. According to the position of fucose in the glycan linkage, fucosylation is divided into core fucosylation and branched fucosylation. FUT can catalyze the biosynthesis of blood group H and Lewis antigens, selectin-mediated leukocyte extravasation, host-pathogen interaction, and modification of signaling pathways, etc.

[0003] Abnormal fucosylation modification plays an important role in the occurrence and development of diseases and can therefore be used as a disease biomarker for diagnosis or prognosis. Abnormal fucosylation is usually closely related to the expression of fucosyltransferase and fucosidase. Many diseases exhibit abnormal changes in fucosylation, including lung cancer, liver cancer, oral cancer, and Alzheimer's disease, etc. For example, abnormal fucosylation of alpha-fetoprotein is approved by the US Food and Drug Administration (FDA) as a biomarker for hepatocellular carcinoma. Therefore, by enriching fucosylated glycoproteins from complex protein samples and detecting fucose structures and expressions, it can be used as a disease-specific biomarker and can also show the progression and prognosis of diseases. Currently, the main methods for enriching fucosylated proteins are lectin enrichment. For example, lectins such as AAL, UEAI, LCA, and AAO are commonly used for enriching fucosylated glycoproteins, but they have disadvantages such as poor lectin method specificity and low sensitivity. Currently, there is a lack of an efficient and convenient method to enrich fucosylated glycoproteins and analyze the structures and abundances of specifically linked fucosylated glycoproteins.

[0004] Therefore, it is necessary to develop a method based on solid-phase fucosylated glycoprotein enrichment and fucosylation cleavage analysis to solve the problem of identifying fucosylated glycoprotein biomarkers in the prior art. Summary of the Invention

[0005] The object of the present invention is to provide a method for solid-phase fucosylated glycoprotein enrichment and fucosylation cleavage analysis.

[0006] One technical solution of the present invention is as follows:

[0007] A method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic cleavage analysis, comprising the steps:

[0008] 1) Extraction of salivary proteins, cellular proteins or tissue proteins;

[0009] 2) Preparation of protein polypeptides and glycopeptides;

[0010] 3) Enrichment of core fucosylated glycoproteins or enrichment of branched fucosylated glycoproteins;

[0011] 4) Mass spectrometry analysis.

[0012] Further, in step 2), the preparation of the protein polypeptides and glycopeptides includes:

[0013] (1) Proteolytic digestion:

[0014] ① Take the protein according to the measured concentration and dissolve it in urea, gently shake until the protein is completely dissolved to obtain a sample;

[0015] ② Add dithiothreitol to the sample and incubate;

[0016] ③ Add iodoacetamide to the sample and incubate in the dark at room temperature;

[0017] ④ Add dithiothreitol to the sample and incubate to quench the remaining unreacted iodoacetamide;

[0018] ⑤ Add HPLC water to the sample to adjust the pH of the sample to 8 - 9;

[0019] ⑥ Add trypsin and incubate overnight to obtain a sample solution;

[0020] (2) Polypeptide purification:

[0021] ① Add trifluoroacetic acid to the sample solution until the pH is adjusted down to 2 - 3;

[0022] ② Add the sample solution to a pre-treated C18 extraction column and repeat the operation multiple times;

[0023] ③ Wash the C18 extraction column with trifluoroacetic acid multiple times;

[0024] ④ Add trifluoroacetic acid to the sample solution until the pH is adjusted down to 2 - 3, repeat multiple times to obtain polypeptides;

[0025] ⑤ Combine the eluted polypeptides and vacuum freeze-dry to obtain a purified polypeptide sample.

[0026] Further, in step (1) ①, the initial concentration of the protein is 6 - 8 μg / μl.

[0027] Further, in step (1)⑤, the concentration of urea in the sample < 1.6M.

[0028] Further, in step (1)⑥, the mass ratio of trypsin to the sample is 1:50 - 1:100.

[0029] Further, in step 3), the enrichment of core fucosylated glycoproteins includes: obtaining glycopeptides containing fucosylation sites or enriching core fucosylated glycopeptides in two ways.

[0030] Further, the obtaining of glycopeptides containing fucosylation sites includes:

[0031] (a) Endo H and Endo F3 digestion: Add GlycoBuffer 3 buffer to the purified polypeptide sample, and then add a mixture of endoglycosidase H and endoglycosidase F3, incubate overnight to obtain a mixture;

[0032] (b) Glycopeptide oxidation and solid-phase binding:

[0033] ① Add sodium periodate to the mixture for light-shielded reaction to obtain an oxidized sample;

[0034] ② Add TFA to the sample;

[0035] ③ Purify the sample with a C18 column to obtain a purified sample;

[0036] ④ Add hydrazide resin to a centrifuge tube, and then pretreat with deionized water, remove the filtrate, and repeat the operation twice to obtain treated hydrazide resin;

[0037] ⑤ React the purified sample with the treated hydrazide resin to obtain a sample;

[0038] (c) Fucosidase digestion: Add GlycoBuffer 1 buffer to the sample, and then add α1-2,4,6 fucosidase, incubate, vortex and centrifuge to collect the liquid, and then elute multiple times with ACN containing TFA and collect the liquid. Combine the collected liquids and vacuum freeze-dry to obtain glycopeptides containing fucosylation sites.

[0039] Further, the enrichment of core fucosylated glycopeptides includes:

[0040] (a) Endo M digestion: Add NH4HCO3 buffer and Endo M enzyme to the purified polypeptide sample, incubate overnight, and vacuum freeze-dry to obtain a sample;

[0041] (b) Hydrophilic interaction chromatography:

[0042] ① Dissolve the sample in ACN containing TFA.

[0043] ② Add the sample to a pretreated Amide-80 chromatographic column and repeat the operation multiple times;

[0044] ③ Wash the extraction column with ACN containing TFA multiple times;

[0045] ④ Elute the sample successively with 60% ACN containing 0.1% TFA, 40% ACN containing 0.1% TFA, and 0.1% TFA, and collect the eluate;

[0046] ⑤ Combine the eluate, freeze-dry it under vacuum to obtain core fucosylated glycopeptides.

[0047] Further, in step 3), the enrichment of branched fucosylated glycoprotein includes:

[0048] (a) α1-2,4,6 fucosidase digestion: Add GlycoBuffer1 buffer to the purified polypeptide sample, then add a mixed enzyme of α1-2,4,6 fucosidase O and galactosidase, incubate overnight to obtain a mixture;

[0049] (b) Glycopeptide oxidation and solid-phase binding:

[0050] ① Add sodium periodate to the mixture for a light-avoiding reaction to obtain an oxidized sample;

[0051] ② Add TFA to the sample;

[0052] ③ Purify the sample with a C18 column to obtain a purified sample;

[0053] ④ Add hydrazide resin to a centrifuge tube, then add deionized water for pretreatment, discard the filtrate, and repeat the operation twice to obtain pretreated hydrazide resin;

[0054] ⑤ React the purified sample with the pretreated hydrazide resin to obtain a sample;

[0055] (c) Fucosidase digestion: Add Tris-HCl to the sample, then add α1-2,3,4 fucosidase, incubate, vortex and centrifuge to collect the liquid, then elute with ACN containing TFA multiple times and collect the liquid, combine the collected liquid, and freeze-dry the sample under vacuum to obtain branched fucosylated glycopeptides.

[0056] Further, in step 4), the mass spectrometry analysis includes: redissolving the fucosylated glycopeptide or core fucosylated glycopeptide or branched fucosylated glycopeptide containing fucosylation sites in ACN containing FA to obtain a sample, subjecting the sample to liquid chromatography-mass spectrometry analysis to obtain first- and second-order mass spectra, with the chromatographic mobile phase being 10-50% ACN, the mass spectrometry energy CE being 26-32, and performing fucosylated protein and fucosylation site analysis using combined bioinformatics software.

[0057] The present invention provides a method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic digestion analysis, which can specifically enrich fucose and analyze fucosylated glycopeptides from complex protein polypeptides, and has wide applications. For example, it can specifically enrich fucosylated glycoproteins from complex protein samples, and can perform qualitative and quantitative analysis, improving the sensitivity and specificity of detecting fucosylated glycoproteins, and is easy to operate; it can be used for analyzing fucosylated glycoproteins and fucosylation sites in any biological sample; through this method, fucosylation-related disease biomarker analysis can be carried out to discover early diagnosis, development, and prognosis biomarkers for diseases; it can be used for high-throughput processing and analysis of samples to improve sample processing efficiency. Brief Description of the Drawings

[0058] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them,

[0059] Figure 1 is a schematic workflow diagram of preparing glycopeptides by solid-phase enrichment of fucosylated glycopeptides in the present invention;

[0060] Figure 2 is a schematic diagram of digestion with Endo H and Endo F3 in the present invention;

[0061] Figure 3 is a schematic diagram of the oxidation of fucose, mannose, and galactose in the present invention;

[0062] Figure 4 is a schematic workflow diagram of digestion with fucosidase in the present invention;

[0063] Figure 5 is a schematic workflow diagram of enriching fucosylated glycopeptides by digestion with Endo M in the present invention;

[0064] Figure 6 is a schematic diagram of digestion with fucosidase and mannosidase in the present invention. Detailed implementation mode

[0065] The present invention has developed a method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic cleavage analysis. Through the specific reaction of endoglycosidase fucosylglycopeptide, fucosylated glycoproteins can be enriched from complex protein samples, and the structure and abundance of fucosylated glycoproteins can be analyzed, which can be used as biomarkers for disease diagnosis.

[0066] To make the above objects, features and advantages of the present invention more obvious and understandable, the technical solutions of the present invention will be further described below with reference to the drawings and embodiments. However, the present invention is not limited to the listed embodiments, and should also include any other well-known changes within the scope of the rights required by the present invention.

[0067] First of all, the so-called "one embodiment" or "embodiment" herein refers to specific features, structures or characteristics that can be included in at least one implementation mode of the present invention. The "in one embodiment" that appears in different places in this specification does not all refer to the same embodiment, nor is it a separate or selectively exclusive embodiment from other embodiments.

[0068] Secondly, the present invention is described in detail using structure diagrams, etc. When detailing the embodiments of the present invention, for the convenience of explanation, the schematic diagrams will be enlarged locally without following the general scale, and the schematic diagrams are only examples, which should not limit the protection scope of the present invention here. In addition, in actual production, a three-dimensional space including length, width and depth should be included.

[0069] Finally, in the present invention, AAL refers to bitter orange leaf lectin; AAO refers to Aspergillus oryzae lectin; UEAI refers to ebony lectin I; LCA refers to lens lectin.

[0070] Example 1

[0071] Please refer to Figure 1 , Figure 1 which is a schematic workflow diagram for the preparation of glycopeptides by solid-phase enrichment of fucosylated glycopeptides in the present invention. As Figure 1 shown, a method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic cleavage analysis (SPFucE) includes the following steps:

[0072] 1. Protein extraction:

[0073] (1) Saliva collection and extraction of saliva proteins

[0074] Collect the saliva of the subjects from 9:00 am to 11:00 am. The subjects are required to avoid eating, drinking, smoking, or using any oral cleaning products for at least one hour before collection. Five minutes before collection, the subjects are required to rinse their mouths with clear water 2-3 times to ensure oral cleanliness. Under non-stimulated conditions, collect the whole natural secreted saliva and place it in a 50 ml centrifuge tube. Collect 5 ml of saliva within 10 minutes. The whole process is carried out on ice. After collection, centrifuge the saliva sample at 4 °C and 12,000 rpm for 25 minutes. Discard the precipitate and collect the supernatant. Add 100X protease inhibitor to the supernatant to obtain salivary proteins.

[0075] Or,

[0076] (2) Cell protein extraction

[0077] Add 10 ul of 100X protease inhibitor and 10 ul of 100X phosphatase inhibitor mixture to 980 ul of RIPA lysis buffer. After washing the cells 3 times with 1X PBS solution, add 1 ml of RIPA lysis buffer mixture. Use a cell scraper to collect the cells into an EP tube (1.5 - 2.0 ml centrifuge tube). Use an ultrasonic crusher to break the cells on ice for 30 s, cool on ice for 30 s, and repeat the operation 4 - 6 times. Centrifuge the EP tube on a centrifuge (4 °C, 12,000 rpm, 15 min), take the supernatant to obtain cell proteins.

[0078] Or,

[0079] (3) Tissue protein extraction

[0080] Take 100 mg of biological tissue and place it in an EP tube. Rinse it 3 times with PBS, add 200 ul of RIPA lysis buffer mixture, and mash the tissue by grinding or other means. Then break it on ice with an ultrasonic crusher for 30 s, cool on ice for 30 s, and repeat the operation 4 - 6 times. Centrifuge the EP tube on a centrifuge (4 °C, 12,000 rpm, 15 min), take the supernatant to obtain tissue proteins.

[0081] Take 2 - 4 ul of protein from (1), (2), or (3), dilute it 5 - 10 times, and use BCA to test the protein concentration.

[0082] 2. Preparation of protein polypeptides and glycopeptides

[0083] (1) Protease digestion

[0084] ① Dissolve 200 ug - 2000 ug of protein according to the measured concentration in 8M urea, and gently oscillate until the protein is completely dissolved to obtain a sample (it is recommended that the initial protein concentration is 6 - 8 ug / ul);

[0085] ② Add 1 / 10 of the total volume of 100 mM dithiothreitol to the sample and incubate at 37 °C for 30 minutes;

[0086] ③ Add 1 / 10 of the total volume of 160 mM iodoacetamide to the sample, and incubate in the dark at room temperature for 30 - 60 min;

[0087] ④ Then add 1 / 10 of the total volume of 100 mM dithiothreitol to the sample, and incubate at 37 °C for 30 minutes to quench the remaining unreacted iodoacetamide;

[0088] ⑤ Add 5 - 10 volumes of HPLC water to the sample (so that the urea concentration in the sample < 1.6 M), and then use freshly prepared 1 M NH4HCO3 to adjust the pH of the solution to 8 - 9;

[0089] ⑥ Add trypsin (Promega, Madison, WI, USA) at a mass ratio of trypsin - sample protein of 1:50 - 1:100, and incubate overnight at 37 °C to obtain a sample solution.

[0090] (2) Polypeptide purification

[0091] ① Add trifluoroacetic acid (TFA, > 99%, w / v) to the sample solution until the pH is adjusted to 2 - 3;

[0092] ② Add the sample solution to a pretreated C18 extraction column and repeat the operation 2 times;

[0093] ③ Wash the extraction column 4 - 6 times with 1 mL of 0.1% trifluoroacetic acid (TFA, 100 μL trifluoroacetic acid added to 100 mL HPLC water);

[0094] ④ Add trifluoroacetic acid (TFA, > 99%, w / v) to the sample solution until the pH is adjusted to 2 - 3, and repeat 2 times to obtain polypeptides;

[0095] ⑤ Combine the eluted polypeptides and vacuum freeze - dry to obtain a purified polypeptide sample.

[0096] 3. Enrichment of core fucosylated glycoproteins or enrichment of branched - chain fucosylated glycoproteins

[0097] (1) Enrichment of core fucosylated glycoproteins

[0098] Method 1: Obtain fucosylated site - containing glycopeptides

[0099] (a) Digest with Endo H and Endo F3 enzymes (please refer to Figure 2 , Figure 2This is a schematic diagram of the digestion with Endo H and Endo F3 in the present invention. In the figure, the solid square represents N-acetylglucosamine, the shaded square represents N-acetylgalactosamine, the solid circle represents galactose, the shaded circle represents mannose, and the solid triangle represents fucose. As Figure 2 shown, Endo H and Endo F3 can hydrolyze the β(1,4) glycosidic bond between two N-acetylglucosamines in the sample glycopeptides with or without core fucosylation. The specific steps of this step are as follows:

[0100] Add 1X GlycoBuffer 3 buffer (the optimal reaction condition is that 20 μg of glycopeptide and 10 μl of 1X GlycoBuffer buffer form a total reaction volume of 10 μl) (New England BioLabs, MA, USA) to the purified polypeptide sample, and then add 10 - 15 U of endoglycosidase H (Endo H) (New England BioLabs, MA, USA) and 10 - 15 U of glycosidase F3 (Endo F3) (New England BioLabs, MA, USA) mixed enzyme (add 10 - 15 U of endoglycosidase H and 10 - 15 U of endoglycosidase F3 per 20 μg of glycopeptide), incubate overnight at 37 °C to obtain a mixture.

[0101] (b) Glycopeptide oxidation and solid-phase binding. (Please refer to Figure 3 .) Figure 3 This is a schematic diagram of the oxidation of fucose, mannose, and galactose in the present invention. Among them, a is fucose, b is mannose, and c is galactose. As Figure 3 shown, the vicinal diol structure of these three monosaccharides can be oxidized to a vicinal dialdehyde structure by sodium periodate. The specific steps of this step are as follows:

[0102] ① Add 10 mM sodium periodate to the mixture and react in the dark for 1 hour to obtain the oxidized sample;

[0103] ② Add 10 times 0.1% TFA to the sample;

[0104] ③ Purify the sample with a C18 column;

[0105] ④ Take 100 - 150 μl of hydrazide resin (Thermo Fisher Scientific, MA, USA), add it to a 1.5 - 2.0 ml centrifuge tube, pretreat it with 450 - 550 μl of deionized water, discard the filtrate, and repeat the operation twice;

[0106] ⑤ React the sample purified in the third step with the hydrazide resin treated in the fourth step at room temperature for 4 h.

[0107] (c) Fucosidase digestion (see Figure 4 , Figure 4 , which is a schematic diagram of the workflow for fucosidase digestion used in the present invention. In the figure, the solid square represents N-acetylglucosamine, the shaded square represents N-acetylgalactosamine, the shaded circle represents mannose, the solid circle -NH-NH2 represents hydrazide resin, and the curvilinear structure represents the peptide segment. As Figure 4 shows, after the vicinal diol of fucose is oxidized to vicinal dialdehyde, it binds to the hydrazide resin. After digestion with α1-2,4,6 fucosidase, the peptide segment containing the fucosylation site can be released from the hydrazide resin.) The specific steps of this step are as follows:

[0108] Add 200 μL of 1X GlycoBuffer 1 buffer to the sample, then add 20 - 30 U of α1-2,4,6 fucosidase (New England BioLabs, MA, USA), incubate at 37 °C for 2 h, vortex centrifuge and collect the liquid. Then elute twice with 50% ACN (0.1% TFA) and collect the liquid. Combine the liquids collected twice, vacuum freeze-dry the sample to obtain the peptide segment containing the fucosylation site.

[0109] Method 2: Enrichment of core fucosylated glycopeptides (see Figure 5 , Figure 5 , which is a schematic diagram of the workflow for enriching fucosylated glycopeptides by Endo M digestion used in the present invention. In the figure, the dark square represents N-acetylglucosamine, the light square represents N-acetylgalactosamine, the solid circle represents galactose, the shaded circle represents mannose, the triangle represents fucose, and the curvilinear structure represents the peptide segment. As Figure 5 shows, Endo M can hydrolyze the β(1,4) glycosidic bond between two N-acetylglucosamines in the glycopeptides of the sample that do not contain core fucosylated glycopeptides.) The specific steps of this step are as follows:

[0110] (a) Endo M digestion

[0111] Add 200 - 250 μL of 25 mM NH4HCO3 (PH: 7 - 9) buffer and 2 μL of Endo M enzyme to the purified polypeptide sample, incubate at 37 °C overnight, and vacuum freeze-dry the sample.

[0112] (b) Hydrophilic interaction chromatography (HILIC)

[0113] ① Dissolve the sample in 500 μL of 80% ACN (0.1% TFA).

[0114] ② Add the sample to a pre-treated Amide-80 chromatographic column (after treating it three times with 1.0 - 1.2 mL of TFA and 80% CAN containing 0.1% TFA respectively), and repeat the operation 2 times;

[0115] ③ Wash the extraction column 3 times (1.0 - 1.2 mL) with 80% ACN containing 0.1% TFA;

[0116] ④ Elute the sample successively with 400 - 500 μL of 60% ACN containing 0.1% TFA, 400 - 500 μL of 40% ACN containing 0.1% TFA, and 400 - 500 μL of 0.1% TFA, and collect the eluate;

[0117] ⑤ Combine the eluates and freeze-dry the sample under vacuum.

[0118] (2) Enrichment of branched fucosylated glycopeptides

[0119] (a) α1-2,4,6 fucosidase digestion

[0120] Add a certain amount of GlycoBuffer 1 buffer (the optimal reaction condition is that 20 μg of glycopeptide forms a total reaction volume of 10 μL with 10 μL of 1X GlycoBuffer buffer) (New England BioLabs, MA, USA) to the purified polypeptide sample obtained in 2(2)⑤, then add 4 - 40 U of α1-2,4,6 fucosidase O (New England BioLabs, MA, USA) (add 4 - 6 U of α1-2,4,6 fucosidase O per 20 μg of glycopeptide) and 70 - 100 U of galactosidase (New England BioLabs, MA, USA) (add 7 - 10 U of galactosidase per 1 μg of glycopeptide) mixed enzymes, incubate overnight at 37 °C to obtain a mixture. (Please refer to Figure 6 , Figure 6 for the schematic diagram of using fucosidase and mannosidase digestion in the present invention. In the figure, the solid square represents N-acetylglucosamine, the shaded square represents N-acetylgalactosamine, the solid circle represents galactose, the shaded circle represents mannose, and the triangle represents fucose. As Figure 6 shown, after hydrolysis by a1-2,4,6 fucosidase and mannosidase, fucosylated glycopeptides containing only a1-3 fucose can be obtained.

[0121] (b) Glycopeptide oxidation and solid-phase binding:

[0122] ① Add 10 mM sodium periodate to the mixture and react in the dark for 1 hour to obtain the oxidized sample;

[0123] ② Add 10 times 0.1% TFA to the sample;

[0124] ③ Purify the sample using a C18 column;

[0125] ④ Take 100 - 150 μl of hydrazide resin (Thermo Fisher Scientific, MA, USA), add it to a 1.5 - 2.0 ml centrifuge tube, pretreat it with 450 - 550 μl of deionized water, discard the filtrate, and repeat the operation twice;

[0126] ⑤ React the sample purified in step ③ with the hydrazide resin treated in step ④ at room temperature for 4 h.

[0127] (c) Fucosidase digestion

[0128] Add 200 μl of 20 mM Tris - HCl (pH = 6.8) to the sample, then add 20 - 30 U of α1 - 2,3,4 fucosidase (Genovis), incubate at 37 °C for 2 h, vortex - centrifuge and collect the liquid. Then elute twice with 50% ACN (0.1% TFA) and collect the liquid. Combine the liquids collected twice, vacuum - freeze - dry the sample to obtain the peptides containing fucosylation sites.

[0129] 4. Mass spectrometry analysis (LC - MS / MS)

[0130] Redissolve the sample of peptides containing fucosylation sites or core - fucosylated glycopeptides or branched - fucosylated glycopeptides enriched in the third step in 50% ACN (0.2% FA);

[0131] Analyze the sample by liquid chromatography - mass spectrometry to obtain the first - order and second - order mass spectra;

[0132] The mobile phase of chromatography is 10 - 50% ACN, the mass spectrometry energy CE is 26 - 32, and use bioinformatics software to analyze fucosylated proteins and fucosylation sites.

[0133] Example 2

[0134] Enrich fucosylated proteins in the saliva or tissues of lung cancer patients and healthy subjects

[0135] Analyze the LC / MS results using MaxQuant software and GlycReSoft software. Fucosylated proteins unique to lung cancer patients and healthy subjects can be identified. After labeling glycopeptides with TMT or IBT, the expression differences of fucosylated proteins between lung cancer patients at different stages and healthy subjects can be quantitatively analyzed to develop appropriate glycoprotein markers, and the specificity and sensitivity for detecting the stages of lung cancer patients can be detected by a simple method.

[0136] Example 3

[0137] Enrichment of fucosylated proteins in lung cancer cells and normal lung epithelial cells

[0138] Fucosylation modification is generally upregulated in different cancer cells. SPFucE is used to identify specific fucosylated proteins between lung cancer cells and normal lung epithelial cells. After treating the cells with fucosylation inhibitors and labeling with TMT or IBT, the changes in fucosylated proteins between lung cancer cells and healthy lung epithelial cells are quantitatively analyzed, and the signal pathways affected by fucosylation are analyzed and identified. Analyzing the upstream and downstream regulatory factors related to the signal pathways is of great significance for identifying potential drug targets in lung cancer in the future.

[0139] Example 4

[0140] Enrichment of fucosylated glycoproteins in bronchoalveolar lavage fluid of lung cancer patients and non-cancerous bronchoalveolar lavage fluid

[0141] Bronchoalveolar lavage fluid is particularly important for studying lung disease markers and discovering disease-related molecules. Bronchoalveolar lavage fluid (BALF) is used to collect samples from the lungs for testing. In this process, a salt solution is passed through a bronchoscope to wash the airways and collect liquid samples. BALF can play an important role in the accurate and confident diagnosis of specific forms of interstitial lung disease. When combined with comprehensive clinical information and high-resolution computed tomography, the nucleated immune cell pattern in BALF can usually provide useful information for diagnostic evaluation and reduce the need for more invasive procedures such as surgical lung biopsy. Bronchoalveolar lavage fluid has been widely used to identify biomarkers of lung diseases. By using the present invention, fucosylated glycoprotein markers related to lung cancer or inflammation in bronchoalveolar lavage fluid can be discovered.

[0142] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic digestion analysis, which is of great significance for discovering disease markers with fucosylation in diseased cells, clinical tissues, and human body fluid specimens, as well as for the research of early disease diagnosis and prognostic markers.

[0143] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic digestion analysis, characterized in that, Including the steps: 1) Extraction of salivary proteins, cellular proteins or tissue proteins; 2) Preparation of protein polypeptides and glycopeptides; 3) Enrichment of core fucosylated glycoproteins or enrichment of branched fucosylated glycoproteins: The enrichment of core fucosylated glycoproteins includes: (a) Endo M digestion: Add NH4HCO3 buffer and Endo M enzyme to the purified polypeptide sample, incubate overnight, and vacuum freeze-dry to obtain a sample; (b) Hydrophilic interaction chromatography: ① Dissolve the sample in ACN containing TFA; ② Add the sample to a pretreated Amide-80 chromatographic column and repeat the operation multiple times; ③ Wash the extraction column multiple times with ACN containing TFA; ④ Elute the sample successively with 60% ACN containing 0.1% TFA, 40% ACN containing 0.1% TFA, and 0.1% TFA, and collect the eluate; ⑤ Combine the eluates and vacuum freeze-dry to obtain core fucosylated glycopeptides; The enrichment of branched fucosylated glycoproteins includes: (a) α1-2,4,6 fucosidase digestion: Add GlycoBuffer 1 buffer to the purified polypeptide sample, and then add a mixed enzyme of α1-2,4,6 fucosidase O and galactosidase, incubate overnight to obtain a mixture; (b) Glycopeptide oxidation and solid-phase binding: ① Add sodium periodate to the mixture for light-avoiding reaction to obtain an oxidized sample; ② Add TFA to the sample; ③ Purify the sample with a C18 column to obtain a purified sample; ④ Add hydrazide resin to a centrifuge tube, pretreat with deionized water, remove the filtrate, and repeat the operation twice to obtain pretreated hydrazide resin; ⑤ React the purified sample with the pretreated hydrazide resin to obtain a sample; (c) Fucosidase digestion: Add Tris-HCl to the sample, then add α1-2,3,4 fucosidase, incubate, vortex centrifuge and collect the liquid, then elute multiple times with ACN containing TFA and collect the liquid, combine the collected liquids, and vacuum freeze-dry the sample to obtain branched fucosylated glycopeptides; 4) Mass spectrometry analysis.

2. The method for solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic cleavage analysis according to claim 1, characterized in that, In step 2), the preparation of protein polypeptides and glycopeptides includes: (1) Proteolysis: ① Take the protein according to the measured concentration and dissolve it in urea, gently shake until the protein is completely dissolved to obtain a sample; ② Add dithiothreitol to the sample and incubate; ③ Add iodoacetamide to the sample and incubate in the dark at room temperature; ④ Add dithiothreitol to the sample and incubate to quench the remaining unreacted iodoacetamide; ⑤ Add HPLC water to the sample to adjust the pH of the sample to 8-9; ⑥ Add trypsin and incubate overnight to obtain a sample solution; (2) Polypeptide purification: ① Add trifluoroacetic acid to the sample solution until the pH is lowered to 2-3; ② Add the sample solution to a pretreated C18 extraction column and repeat the operation multiple times; ③ Wash the C18 extraction column multiple times with trifluoroacetic acid; ④ Add trifluoroacetic acid to the sample solution until the pH is adjusted to 2-3, and repeat multiple times to obtain polypeptides. ⑤ Combine the eluted polypeptides and vacuum freeze-dry to obtain a purified polypeptide sample.

3. A method for solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic digestion analysis according to claim 2, characterized in that: In step (1) ①, the initial concentration of the protein is 6-8 ug / ul.

4. A method for solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic cleavage analysis according to claim 2, characterized in that: In step (1) ⑤, the concentration of urea in the sample is <1.6 M.

5. A method for solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic digestion analysis according to claim 2, characterized in that: In step (1) ⑥, the mass ratio of trypsin to the sample is 1:50 - 1:

100.

6. A method for solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic digestion analysis according to any one of claims 1 to 5, characterized in that In step 4), the mass spectrometry analysis includes: redissolving the core fucosylated glycopeptide or branched fucosylated glycopeptide in ACN containing FA to obtain a sample, subjecting the sample to liquid chromatography-mass spectrometry analysis to obtain primary and secondary mass spectra, with the chromatographic mobile phase being 10-50% ACN, the mass spectrometry energy CE 26-32, and performing fucosylated protein and fucosylation site analysis using bioinformatics software.

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