GlycoDepth method for improving identification depth of glycoprotein group and application of GlycoDepth method

Abstract

The invention provides a GlycoDepth method for remarkably improving the identification depth of a glycoprotein group by utilizing a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9) gene editing technology and an inhibitor treatment material and combining site-specific N-glycoprotein omics analysis. According to the method, enrichment of specific glycoforms in plant materials such as tomatoes is realized through targeted knockout or inhibition of specific glycosyl processing enzymes, so that heterogeneity of glycopeptides is reduced, and the sensitivity of mass spectrometric detection and the identification depth of N-glycoprotein groups are improved. The invention also provides application of the method in glycoprotein function research, crop quality improvement and sugar engineering.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and glycoproteomics, specifically relating to the GlycoDepth method and its application, which enriches specific glycoforms through CRISPR / Cas9 gene editing and inhibitor treatment, thereby improving the depth of glycoproteome identification. Background Technology

[0002] Protein glycosylation is a process in which glycans covalently bind to specific amino acid sequences of proteins. It is a widespread and important post-translational modification (Schjoldager KT, Narimatsu Y, Joshi HJ, Clausen H. Globalview of human protein glycosylation pathways and functions. Nat Rev Mol CellBiol. 2020;21(12):729-749.). It mainly includes two types: N-glycosylation and O-glycosylation. N-glycosylation is the main form of protein glycosylation modification and has a profound impact on protein function, stability, subcellular localization, and other properties (Krzysztof M, Radoslaw K, Marcin C. How glycosylation affects glycosylation: the role of N-glycans in glycosyltransferase activity[J]. Glycobiology,2020.). Glycoproteins account for more than 50% of total cellular proteins and participate in many important life processes such as immune response, reproduction, pathogen infection, and cancer. The Human Glycome Atalas Project (HGA) was officially launched in April 2023, and protein glycosylation research has become an important scientific frontier in the post-genomic era.

[0003] Research tools are a crucial foundation for glycosylation decoding. Methods used for protein glycosylation measurement mainly include mass spectrometry, nuclear magnetic resonance (NMR), and chromatography (Fontana C, Widmalm G. Primary Structure of Glycans by NMR Spectroscopy. 2023.). Compared with other techniques, mass spectrometry offers numerous advantages such as high sensitivity, high throughput, and high resolution, making it an important tool in glycoproteomics research. Currently, N-glycoproteomics analysis based on biomolecular mass spectrometry is mainly conducted at three levels: intact glycoproteins, glycosylated peptides, and site-specific intact glycopeptides. With advancements in glycopeptide enrichment materials, mass spectrometry hardware, data analysis software, and glycobioinformatics technologies, protein glycosylation mass spectrometry analysis has made some progress. However, the following problems still exist: the high heterogeneity of protein glycosylation caused by a series of enzymatic catalysis poses technical challenges to the detection efficiency of intact glycopeptides and the analytical depth of glycoproteomics.

[0004] Glycoengineering is an important tool for studying protein glycosylation function. It utilizes gene editing, enzyme engineering, and chemical methods to regulate the structure, synthetic pathways, and function of glycans (Narimatsu Y, Bull C, Yang Z, et al. Genetic glycoengineering in mammalian cells. Glycobiology. 2021(12):31.). Compared to enzyme engineering and chemical methods, CRISPR / Cas9 gene glycoengineering technology offers advantages such as precise targeting, strong design capabilities, good plasticity, and cross-species compatibility. By targeting and editing the genes encoding glycan processing enzymes, gene glycoengineering technology can design and programmatically modify protein glycans.

[0005] The widespread distribution and significant biological functions of protein glycosylation have solidified its importance. Improving the depth of glycoproteome identification is of significant methodological and scientific importance. While technological advancements have led to substantial improvements in glycopeptide enrichment materials and mass spectrometry hardware, the lack of cost-effective commercially available glycopeptide enrichment materials and the high costs associated with upgrading mass spectrometry hardware limit the feasibility of in-depth glycoproteome identification.

[0006] In summary, existing technologies lack a universal method to overcome the high heterogeneity of glycopeptides and improve the depth of glycoproteome identification. Summary of the Invention

[0007] To address the problems existing in current technologies, this invention provides a GlycoDepth method and its application for improving the depth of glycoproteomics identification. Specifically, this invention aims to provide a GlycoDepth method that significantly improves the depth of glycoproteomics identification by enriching specific glycoforms through CRISPR / Cas9 gene editing and inhibitor treatment, combined with site-specific N-glycoproteomics analysis. First, using CRISPR / Cas9 gene editing technology and the Kif base (a specific inhibitor of the protein N-glycosylation processing enzyme α-ManI), gene knockout mutants and inhibitor-treated materials enriched with specific glycoforms Man8GlcNAc2 and Man9GlcNAc2 are customized. Site-specific N-glycoproteomics technology is used to characterize and determine the enrichment characteristics of specific glycoforms at the intact glycopeptide level in the obtained tomato materials. Furthermore, the site-specific N-glycoproteomics system is used to systematically analyze the effect of gene knockout mutants and inhibitor-treated materials on improving the depth of glycoproteomics identification, proposing the underlying principles and methodological advantages of GlycoDepth. In materials enriched with specific glycoforms, the heterogeneity of glycopeptides is significantly reduced, thus greatly improving the detection sensitivity and coverage of glycopeptides by mass spectrometry.

[0008] The technical solution of the present invention is as follows: This invention provides a GlycoDepth method for improving the depth of glycoproteome identification, characterized by comprising the following steps: a) Obtaining treated material enriched with specific glycoforms by treating biological material in at least one of the following ways: (1) Using the CRISPR / Cas9 system, mutants of the gene encoding glycosyl processing enzymes were obtained; (2) Use specific inhibitors of glycosyl processing enzymes; b) Perform site-specific glycoproteomics analysis on the treated material.

[0009] Furthermore, the GlycoDepth method combines glycoengineering techniques to directionally modify and enrich specific glycoforms and site-specific glycoproteins; Furthermore, the GlycoDepth method is a general method for improving the depth of glycoproteome identification; Furthermore, the glycoprotein group refers to all types of glycosylation that exhibit site heterogeneity, including N-glycosylation; Furthermore, the identification refers to identification at the glycosylation site, glycopeptide, and glycoprotein levels; Furthermore, the identification depth refers to the number of glycosylation sites, glycopeptides, and glycoproteins identified; In some embodiments, the glycosyl processing enzyme is a protein N-glycosylation processing enzyme or an O-glycosylation processing enzyme. Preferably, the protein glycosyl processing enzyme is an N-glycosylation processing enzyme. More preferably, the N-glycosylation processing enzyme is an α-mannosidase. Most preferably, the α-mannosidase is a type I α-mannosidase.

[0010] In some embodiments, the gene encoding glycosylation processing enzyme is a gene encoding protein glycosylation processing enzyme; more preferably, the gene encoding protein glycosylation processing enzyme is a gene encoding N-glycosylation processing enzyme; most preferably, the gene encoding N-glycosylation processing enzyme is SlMNSI1; preferably, the gene encoding glycosylation processing enzyme includes non-redundant genes responsible for encoding glycosylation processing enzymes, including SlMNSI1.

[0011] In some embodiments, the specific glycotype is a type of glycan chain modified by protein glycosylation, including at least one of N-glycan chains and O-glycan chains. Preferably, the N-glycan chain is a high-mannose N-glycan chain, and more preferably, the high-mannose N-glycan chain is at least one of Man8GlcNAc2 and / or Man9GlcNAc2.

[0012] In some embodiments, the specific inhibitor is kifunensine.

[0013] In some embodiments, the biomaterial is plant, animal, or microbial tissue; preferably, the biomaterial is plant material, more preferably, the plant material is fruit organoids, and most preferably, the fruit organoids are tomato fruits.

[0014] In some embodiments, the mutant is the inactivation or downregulation of a gene encoding a glycosyl processing enzyme.

[0015] In some embodiments, the glycopeptide heterogeneity of the treated material is reduced.

[0016] In some embodiments, the method is based on slmnsI1 The mutant specifically enriched Man8GlcNAc2, and treatment with the Kif inhibitor specifically enriched Man9GlcNAc2.

[0017] This invention provides an application of the method described above in glycoprotein function research, crop quality improvement, or glycoengineering.

[0018] This invention provides a CRISPR / Cas9 gene editing vector for implementing the above method, characterized in that it targets a glycosyl processing enzyme gene.

[0019] This invention also provides a method for preparing complete N-glycopeptides applicable to various tissues and parts of plants. This invention also provides a step-collision energy / high-energy collision dissociation (SCEHCD) mass spectrometry method suitable for the identification of intact glycopeptides. Attached Figure Description Figure 1 The graph shows the effect of kifuminensine (Kif) inhibition of α-1,2-mannosidase on tomato fruit development and the accumulation of Man9GlcNAc2(H9N2F0X0); (a) shows the fruit phenotypes of the Mock control group and the Kif treatment group at 29, 32, and 38 days after flowering (DPA); the scale bar represents 1 cm; (b) shows the fruit weights of the Mock control group and the Kif treatment group at 29, 32, and 38 days after flowering (DPA); (c) shows the seed germination rates of the Mock control group and the Kif treatment group; seeds were separated from fruits 3 days after color breaking (Br+3); (d) shows the composition and content of N-glycans in tomato fruits at the color breaking stage (Br) as detected by MALDI-TOF-MS; (e) shows the color breaking stage. MALDI-TOF-MS mass spectra of total N-glycans in fruits from the color-changing stage (Br) Mock control group and Kif treatment group; (f) phylogenetic tree analysis of MNSI family members in tomato and Arabidopsis thaliana; (g) RT-qPCR analysis of SlMNSI gene expression levels in immature green (IMG), mature green (MG), color-breaking stage (Br), and red ripe stage (RR) tomatoes; Monosaccharide composition is represented as: H#N#F#X#, where H, N, F, and X represent the number of residues of hexose, N-acetylglucosamine (HexNAc), fucose, and xylose, respectively; Monosaccharide structure symbols: green circle represents mannose; blue square represents N-acetylglucosamine; red triangle represents fucose; gray pentagram represents xylose.

[0020] Figure 2 To knock out SlMNSI1 Diagram showing the accumulation of Man8GlcNAc2(H8N2F0X0) leading to multiple developmental defects in tomatoes; (a) is a homozygous mutant. slmnsI1 CRISPR / Cas9 editing site sequence analysis; editing sites and editing types (insertion or deletion) are marked in blue; (b) Seed size 3 days after germination (DPG); (c) Plant phenotype 30 days after germination (DPG); (d) Leaf phenotype 30 days after germination (DPG); (e) Root phenotype 30 days after germination (DPG); (f) Fruit setting phenotype 90 days after germination (DPG); (g) Fruit phenotype 30, 34, 37, 40, and 43 days after flowering (DPA); Scale bar represents 5 cm (applicable to b–g); (h) Color breaking stage (Br) wild-type (WT) and mutant. slmnsI1MALDI-TOF-MS mass spectra of total N-glycan chains in fruit; (i) Composition and content of N-glycans in tomato fruit at the color-breaking stage as determined by MALDI-TOF-MS; Monosaccharide composition is expressed as: H#N#F#X#, where H, N, F and X represent the number of residues of hexose, N-acetylglucosamine (HexNAc), fucose and xylose, respectively; Monosaccharide structure symbols: green circle represents mannose; blue square represents N-acetylglucosamine; red triangle represents fucose; gray pentagram represents xylose; Figure 3 Treatment groups and mutants with kifunensine (Kif) slmnsI1 The enrichment effect of specific sugar forms on N-glycopeptides in tomato fruit is shown in the figure; (a) the percentage of four types of N-glycopeptides in the Mock control group and the Kif treatment group; (b) the percentage of N-glycopeptides modified by Man9GlcNAc2(H9N2F0X0) in the Mock control group and the Kif treatment group; (c) wild type (WT) and mutant slmnsI1 Percentage of the four classes of N-glycopeptides; (d) wild-type (WT) and mutant slmnsI1 The percentage of N-glycopeptides modified by Man8GlcNAc2(H8N2F0X0); monosaccharide structure symbols: green circles represent mannose; blue squares represent N-acetylglucosamine.

[0021] Figure 4 The diagram shows the site-specific N-glycoproteomics analysis; (a) compared with the previously reported N-glycoproteome of tomato fruit, the GlycoDepth method identified more N-glycopeptides (3091), N-glycosylation sites (873), N-glycoproteins (573), and N-glycoforms (158); (b) conserved motif analysis of the 873 identified N-glycosylation sites; classic NXS / T sequences were significantly enriched; (c) Mock control group (injected with ddH2O), Kif treatment group, wild type (WT) (uninjected), and mutant. slmnsI1 Principal component analysis (PCA) of intact N-glycopeptides in fruit during the color-breaking stage.

[0022] Figure 5 Figure 1 shows the principle of the GlycoDepth method and the effect of improving the identification depth; (a) shows the principle of GlycoDepth in improving the identification depth of N-glycoproteins; specific glycoforms on glycopeptides (e.g., Man9GlcNAc2 and mutants in Kif-treated fruits). slmnsI1Enrichment of Man8GlcNAc2 in the fruit reduced the heterogeneity and complexity of intact N-glycopeptides, and essentially had a significant enrichment effect on the detection of glycosylation sites, which is more conducive to the detection of glycosylation sites with low abundance, thereby improving the identification depth of glycoproteins (sacrificing the number of glycoforms to improve the overall identification depth of glycosylation); (b) shows the improvement in the number of N-glycopeptides, N-glycosylation sites and N-glycoproteins identified in the Kif treatment group compared with the Mock control group; (c) shows the improvement in the number of mutant N-glycopeptides, N-glycosylation sites and N-glycoproteins compared with the wild-type (WT) group. slmnsI1 The effect of increasing the number of identified N-glycopeptides, N-glycosylation sites, and N-glycoproteins. Detailed Implementation

[0023] The technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] The terms used in this invention are explained as follows: In this invention, the sugar chain structure is represented by the format "HnNmFaXb", wherein: H represents hexose residues (mainly mannose / galactose / glucose) in galactose or high-mannose structures, and the following number n indicates the number of hexose residues. N represents N-acetylglucosamine residues (GlcNAc), and the following number m indicates the number of residues; F represents fucose residues, and the following number 'a' indicates the number of residues. X represents xylose residues, and the following number b indicates the number of residues.

[0025] Therefore, the relevant terms in this invention are explained as follows: H9N2F0X0 indicates that the sugar chain consists of 9 hexoses and 2 N-acetylglucosamines, and does not contain fucose or xylose; H3N2F1X1 indicates that the sugar chain consists of 3 hexose residues, 2 N-acetylglucosamine residues, 1 fucose residue, and 1 xylose residue; H3N3F1X1 indicates that the sugar chain consists of 3 hexose residues, 3 N-acetylglucosamine residues, 1 fucose residue, and 1 xylose residue; H3N4F1X1 indicates that the sugar chain consists of 3 hexoses, 4 N-acetylglucosamines, 1 fucose, and 1 xylose.

[0026] In this invention, the terms "control group," "Mock group," and "Mock control group" all refer to control groups; and the terms "treatment group," "Kif group," and "Kif treatment group" all refer to treatment groups.

[0027] Example 1: Treatment of tomato fruits with kifuninsine (Kif) inhibitors In the early stage of tomato fruit development (17 days after flowering), 50 μL of a 1 mM Kif aqueous solution was injected, while the control group was injected with the same volume of ddH2O. Tomato plants in both groups were cultivated and managed under identical growing conditions, and phenotypes were observed. Fruits in the control group broke color 38 days after flowering, while fruits in the inhibitor-treated group broke color earlier, 32 days after flowering. Figure 1 a). 38 days after flowering, the average single fruit weight in the inhibitor-treated group was significantly lower than that in the control group ( Figure 1 b). In the inhibitor-treated group, the fruits at the Br+6 stage changed color normally, but the seeds inside could not germinate normally. Figure 1 c).

[0028] Fruits from the same developmental stage (Breaker stage) in both the control and treatment groups were used as materials for the extraction, purification, and preparation of total N-glycans. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI) was then used to detect the total N-glycans and analyze the qualitative and quantitative changes in each sugar form. The results showed that the N-glycans in both groups were mainly concentrated in the nucleus-to-cytoplasm ratio (N / C). m / z The range is from 1200 to 2200. Figure 1 The N-glycans in the control group were... m / z The highest peak value was observed at 1365.432, indicating that the N-glycans in the treatment group were at... m / z The highest peak value was found at 2059.759, indicating that the N-glycan processing in the fruit of the treatment group was blocked. Figure 1 Using the GlycoMod website and Bruker Flexanalysis 3.4 software to analyze the structure and content of N-glycans, it was found that in the tomato fruits of the treatment group, the high-mannose type N-glycan Man9GlcNAc2(H9N2F0X0) accumulated in large quantities, significantly higher than that in the control group, while the sugar forms of Man3GlcNAc2Fuc1Xyl1(H3N2F1X1), Man3GlcNAc3Fuc1Xyl1(H3N3F1X1), and Man3GlcNAc4Fuc1Xyl1(H3N4F1X1) were significantly lower than those in the control group. Figure 1 ).

[0029] Example 2: SlMNSI1 CRISPR / Cas9 gene knockout and mutant acquisition Designing targets using CRISPR-P online tools SlMNSI1 The sgRNA of the gene was used to construct the pYLCRISPR / Cas9Pubi-H-SlMNSI1 vector, which was then transformed into tomato cotyledon explants via Agrobacterium-mediated transformation. Homozygous mutant lines without Cas9 were obtained by PCR screening. slmns1#7 and slmnsI1#15 In these two homozygous lines, SlMNSI1 The specific editing methods of gene targets are shown in the peak diagram. Figure 2 As shown in a. slmnsI1#7 The homozygous mutant inserts 1 bp at the target site. slmnsI1#15 The homozygous mutants lacked a 1 bp target site. The SlMNSI1 protein in these two homozygous lines experienced a frameshift effect due to the base mutation, causing premature termination of translation and ultimately resulting in truncated proteins containing only 23 and 19 amino acid residues, respectively, leading to the complete loss of the GH47 glycosyl hydrolase functional domain. The wild-type (WT) and mutant (…) slmnsI1 Tomato plants were cultivated and managed under uniform growing conditions, and their phenotypes were observed. slmnsI1 The growth and development of the mutant plants were significantly affected. Thirty days after flowering... slmnsI1 The mutant plants exhibited stunted growth; 90 days after flowering, slmnsI1 The mutant exhibits poor leaf growth and a low fruit set rate in the first spike. Figure 2 (b-2f). In addition... slmnsI1 The mutant fruit was significantly smaller than the WT fruit and broke color 9 days earlier. Figure 2 g).

[0030] With WT and mutants slmnsI1 Fruits at the same developmental stage, such as the breakaker stage, were used as materials for the extraction, purification, and preparation of total N-glycans. Further MALDI analysis of the total N-glycans was performed to analyze the qualitative and quantitative changes in each sugar form. The results showed that the N-glycans in WT (whole taurine)... m / z The highest peak value is at 1365.463, indicating that the main N-glycoform in WT is H3N2F1X1 ( Figure 2 ). slmnsI1#7 and slmnsI1#15 The mutant's N-glycan chain in m / z The highest peak value is at 1897.874, indicating that the main N-glycan in the mutant is Man8GlcNAc2(H8N2F0X0). The N-glycan processing in the mutant is blocked at H8N2F0X0, preventing further processing and synthesis of the unique N-glycan structure. This leads to a global impact on fruit growth, development, and the metabolic network. Figure 2 ).

[0031] Example 3: Preparation of total N-glycans and analysis by matrix-assisted laser desorption / ionization mass spectrometry (MALDI) (1) Total protein extraction: Weigh 1 g of fruit sample into a 10 mL centrifuge tube, add 3 mL of Buffer Y buffer (1 M Tris-HCl, 10% SDS, 0.5 M EGTA, 0.5 M EDTA, β-mercaptoethanol, 0.1 M PMSF, 100 Protein inhibitor), vortex thoroughly, and then place in a 65 ℃ water bath for 15 min to allow for complete lysis. Centrifuge at 12000 rpm at room temperature for 30 min, and transfer the supernatant to a new 10 mL centrifuge tube. Add 3 mL of cold phenol and vortex thoroughly. Centrifuge at 12000 rpm at 4 ℃ for 15 min, discard the supernatant, add 3 mL of Buffer Z buffer (0.05 M Tris-HCl), and vortex thoroughly. Centrifuge at 12000 rpm at 4 ℃ for 15 min, discard the supernatant, add 15 mL of 0.1 M ammonium acetate methanol solution, and precipitate overnight at -20 ℃. The following day, the sample was centrifuged again for 20 minutes, and the white precipitate was washed twice each with methanol and acetone. The resulting white precipitate was the crude protein of the sample.

[0032] (2) Enzymatic hydrolysis of proteins: The crude protein precipitate extracted from the sample was dissolved in 3 mL Tris-HCl (0.01 M, pH=8). After denaturing the protein at 100 ℃, 2 μL chymotrypsin (30 mg / mL), 2 μL trypsin (30 mg / mL) and 2 μL CaCl2 (0.2 M) were added respectively. The mixture was shaken in a water bath at 37 ℃ for 16 h to hydrolyze the crude protein into peptides. The reaction was terminated by heating at 100 ℃ for 5 min. (3) PNGase A digestion of N-glycans: Centrifuge at 12000 rpm for 20 min at room temperature. Take an equal amount of 1 mg of polypeptide supernatant from each sample, evaporate to dryness under vacuum, add 1 mL of citrate buffer (0.1 M, pH=4) and 0.5 μL of PNGase A (Glycoamidase A), react in a water bath at 37 ℃ for 24 h, and terminate the reaction by heating at 100 ℃ for 5 min. Centrifuge at 12000 rpm for 10 min at room temperature, and take the supernatant for purification.

[0033] (4) Enrichment and purification of N-glycans: First, N-glycans were purified using a C18 solid-phase extraction cartridge (C18 cartridge Supelclean™ ENVI-18, Waters) to remove impurity peptides, proteins, and other contaminants; then, N-glycans were purified using a graphitized carbon solid-phase extraction cartridge (ENVI-Carb™, Supelco) to remove salts and small polar compounds. The purified N-glycan samples were evaporated to dryness by rotary evaporation and then reconstituted in 25 μL of ultrapure water for MALDI mass spectrometry detection.

[0034] (5) MALDI mass spectrometry detection: Glycan detection was performed using a sinapic acid (SA) / 2-hydrazinophthalic acid (2-HTA) composite matrix. The purified N-glycan sample and matrix solution were sequentially mixed on a MALDI target plate, and the derivatization reaction was carried out directly on the target plate. Measurements were performed in negative ion reflectance mode on an UlrafleXtreme™ MALDI-TOF / TOF MS instrument equipped with a Smartbeam Nd:Y AG (355 nm) laser. A 384 polished target plate was used, and mass spectrometry data analysis was performed using Bruker Flexanalysis 3.4 software.

[0035] The N-glycan composition structure was identified using GlycoMod (https: / / web.expasy.org / glycomod / ), and the content percentage of each sugar type in the sample was calculated.

[0036] Example 4: Preparation of intact N-glycopeptides and their step-collision energy / high-energy collision dissociation (sceHCD) mass spectrometry analysis (1) Desalting of peptides: The peptides obtained from the enzymatic hydrolysis in Example 3 were desalted using a hydrophilic-lipophilic co-equilibrium (HLB) solid-phase extraction column. This included the following steps: 1) Activation and equilibration: Add 3 mL of chromatographic methanol, 3 mL of 5% acetonitrile (0.1% TFA), 3 mL of 50% acetonitrile (0.1% TFA), and 6 mL of 5% acetonitrile (0.1% TFA) solution in sequence.

[0037] 2) Sample loading: Add peptide solution, collect the effluent, and repeat the sample loading operation once.

[0038] 3) Impurity removal: Add 6 mL of 5% acetonitrile (0.1% TFA) solution, control the impurity removal solution to flow out slowly by gravity, and then carefully dry the impurity removal solution using a vacuum pump. 4) Elution: First add 1 mL of 50% acetonitrile (0.1% TFA) solution, then add 1 mL of 80% acetonitrile (0.1% TFA) solution, and collect the eluent. 5) Evaporation to dryness: The combined eluents were evaporated to dryness at 30°C by rotation, with time controlled. The evaporated peptide sample was then quickly removed and reconstituted in 2 mL of 80% (1% TFA) solution. (2) Enrichment of intact N-glycopeptides The peptides, after desalting via an HLB column, were added to a self-made HILIC solid-phase extraction column (containing 100 mg of Agela Technologies resin particles) for purification of intact glycopeptides, specifically including the following steps: 1) Activation and equilibration: The HILIC column was activated and equilibrated sequentially with ddH2O (1% TFA) and 80% acetonitrile / 1% trifluoroacetic acid (TFA) solution; 2) Sample loading: Load the intact N-glycopeptides after HLB desalting, collect the effluent, and repeat the sample loading operation once. 3) Impurity removal: Add 1 mL of 80% acetonitrile (1% TFA) solution to remove impurities, repeat three times, and then carefully dry the impurity removal solution using a vacuum pump; 4) Elution: Elute the glycopeptides sequentially with 3 mL of 40% acetonitrile / 1% TFA solution and then with 3 mL of 10% acetonitrile / 1% TFA solution. 5) Evaporation: The combined effluent was evaporated at 30°C by rotation, and the time was controlled. The evaporated peptide sample was quickly taken out and reconstituted in 20 μL of 1% TFA solution and stored at -80°C.

[0039] (3) Liquid chromatography-mass spectrometry analysis of N-glycopeptides (nLC-ESI-MSn) Intact N-glycopeptides dissolved in 0.1% TFA aqueous solution were analyzed by nLC-ESI-MS / MS on a Q Exactive HF mixed mass spectrometer (Thermo Scientific) with a Nanospray Flex ion source and an Ultimate 3000 nm UPLC system (Thermo Scientific). Samples were loaded onto a pre-column (PepMap C18, 100A, 100 μm × 2 cm, 5 μm, Thermo Scientific) and separated on the analytical column (PepMap C18, 100A, 75 μm × 50 cm, 2 μm, Thermo Scientific) at a flow rate of 300 nL / min. Solvent A was 0.1% formic acid aqueous solution, and solvent B was 0.1% formic acid in acetonitrile solution. Chromatographic gradient: Solvent B increases from 5% to 8% within 6 minutes, from 8% to 20% within 137 minutes, from 20% to 40% within 73 minutes, then increases to 90% within 5 minutes and maintains an isocratic gradient for 8 minutes, and finally decreases to 5% within 6 minutes.

[0040] Mass spectrometry parameters: MS1 scan range is... m / z From 800 to 2000, secondary mass spectrometry (MS2) was performed using a stepped collision energy / high-energy collision dissociation (sceHCD) fragmentation mode, with collision energies set to 20%, 30%, and 40%, respectively.

[0041] Raw data analysis of intact glycopeptides: pGlyco3 was used for qualitative identification of glycopeptides from raw mass spectrometry data, and pGlycoQuant was used for quantitative analysis of glycopeptides.

[0042] Example 5: Kif-treated materials and slmnsI1 Enrichment effect of specific glycoforms in mutants Based on N-glycomic characterization ( Figure 1 and Figure 2 The site-specific N-glycoproteomics technique was used to further characterize and determine the enrichment characteristics of specific glycotypes at the level of intact glycopeptides in the obtained tomato materials. Figure 3 The results showed that, compared with the Mock group (control group), the Kif group (treatment group) had a significant increase in high-mannose N-glycopeptides, while complex and oligomannose N-glycopeptides were significantly reduced. Figure 3 a). The average proportion of high-mannose N-glycopeptides significantly increased from 24.96% in the Mock group to 74.49% in the Kif group ( Figure 3 a). The average percentage of Man9GlcNAc2-modified N-glycopeptides increased from 4.14% in the Mock group to 48.28% in the Kif group. Figure 3 b). Similarly, compared to the wild type (WT), slmnsI1 The mutant showed a significant increase in high-mannose N-glycopeptides and a significant decrease in complex and oligomannose N-glycopeptides. Figure 3 c). The average proportion of high-mannose N-glycopeptides significantly increased from 36.81% in the WT group to 92.63% in the slmnsI1 mutant. Figure 3 c). The average percentage of Man8GlcNAc2-modified N-glycopeptides significantly increased from 12.45% in the WT group to slmnsI1 49.09% of the mutants ( Figure 3 d).

[0043] Example 6: Site-Specific N-Glycoproteomics Analysis Based on the determination of specific glycopeptide accumulation in Example 5, qualitative and quantitative analysis of intact glycopeptides was carried out. Figure 4We identified a total of 3091 N-glycopeptides, corresponding to 573 N-glycoproteins, 873 N-glycosylation sites, and 158 N-glycoforms. Compared with previously reported tomato N-glycoproteomes (Ruiz-May et al., 2014; Zhang et al., 2020), this study achieved the most comprehensive tomato N-glycoproteome analysis to date. Figure 4 a).

[0044] Treatment group using inhibitors and slmnsI1 The enrichment characteristics of specific glycoforms in mutants can be significantly enhanced. Sequence analysis shows that the vast majority of identified N-glycosylation sites conform to the classic Asn-X-Ser / Thr motif (…). Figure 4 b). Principal component analysis (PCA) was performed on the 3091 detected intact N-glycopeptides. The results showed good reproducibility among biological replicates and clear differentiation between samples. Figure 4 c). The above results demonstrate that the site-specific N-glycoproteomics analysis provided in this embodiment is reliable.

[0045] Example 7: Principle of GlycoDepth Method and its Effect on Improving Identification Depth Based on the above embodiments, we propose the GlycoDepth method to improve the depth of glycoprotein identification. Its main principle is that glycoengineering techniques based on CRISPR / Cas9 gene editing or inhibitor treatment can target and customize glycoform modifications on proteins, reducing the heterogeneity of glycoform modifications and achieving a significant enrichment effect. Under the same chromatographic and mass spectrometric detection conditions, this can significantly improve the detection depth of glycoproteins. Figure 5 a). Treatment group using inhibitors and slmnsI1 Mutant materials significantly improve the depth of glycoproteome identification. Figure 5 b and Figure 5 c).

[0046] Industrial applicability The CustomGlyco method provided by this invention can be widely applied to glycobiology research, crop quality improvement, glycoengineering, and pharmaceutical fields in different species, and has high versatility and practicality.

[0047] Those skilled in the art will understand that the steps, measures, and schemes in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted; furthermore, other steps, measures, and schemes in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted; furthermore, the steps, measures, and schemes in the prior art that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above-described embodiments are merely illustrative of several implementation methods of this disclosure, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the patent for the embodiments of this disclosure. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the embodiments of this disclosure, and these all fall within the protection scope of the embodiments of this disclosure. Therefore, the protection scope of the embodiments of this disclosure should be determined by the appended claims. As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and detail can be made without departing from the spirit and scope of the present invention as defined in the appended claims.

[0048] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, such designs should fall within the protection scope of the present invention.

Claims

1. A GlycoDepth method for improving the depth of glycoprotein panel identification, characterized in that, The method comprises the following steps: a) obtaining a treatment material with enriched specific glycoforms by treating a biological material by at least one of the following ways: (1) using CRISPR / Cas9 system to obtain a mutant of a gene encoding a glycosyl processing enzyme; (2) using a specific inhibitor of a glycosyl processing enzyme; b) performing site-specific glycoproteomics analysis on the treatment material.

2. The method of claim 1, wherein, The glycosyl processing enzyme is a protein N-glycosylation processing enzyme or an O-glycosylation processing enzyme, preferably, the protein glycosyl processing enzyme is an N-glycosylation processing enzyme, more preferably, the N-glycosylation processing enzyme is an α-mannosidase, most preferably, the α-mannosidase is a type I α-mannosidase.

3. The method of claim 1, wherein, The gene encoding the glycosyl processing enzyme is a gene of a protein glycosylation processing enzyme, more preferably, the gene of the protein glycosylation processing enzyme is a gene of an N-glycosylation processing enzyme, most preferably, the gene of the N-glycosylation processing enzyme is SlMNSI1; preferably, the gene encoding the glycosyl processing enzyme includes non-redundant genes responsible for encoding glycosyl processing enzymes including SlMNSI1.

4. The method of claim 1, wherein, The specific glycoform is a type of sugar chain of protein glycosylation modification, including at least one of N-glycan and O-glycan, preferably, the N-glycan is a high mannose type N-glycan, more preferably, the high mannose type N-glycan is Man8GlcNAc2 and / or Man9GlcNAc2.

5. The method of claim 1, wherein, The specific inhibitor is kifunensine.

6. The method of claim 1, wherein, The biological material is plant, animal or microbial tissue; preferably, the biological material is plant material, more preferably, the plant material is fruit-like organ, most preferably, the fruit-like organ is tomato fruit.

7. The method of claim 1, wherein, The mutant is inactivation or down-regulation of expression of the gene encoding the glycosyl processing enzyme.

8. The method of claim 1, wherein, The glycopeptide heterogeneity of the treatment material is reduced.

9. Use of the method according to any one of claims 1-8 in glycoprotein function research, crop quality improvement or glycoengineering.

10. A CRISPR / Cas9 gene editing vector for implementing the method of claim 1, characterized in that, Targeting the glycosyl processing enzyme gene.

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