Method for marking core fucosylation and / or O-GlcNAc glycosylation

Abstract

The invention provides an enrichment method which comprises the following steps: (s1) carrying out ligation reaction on a temperature-sensitive capture agent and a substrate to be captured under an enzyme catalysis condition; (s2) adjusting the salt concentration, separating the precipitate, and dissolving to obtain a compound containing'temperature-sensitive trapping agent-substrate '; (s3) releasing the substrate and the temperature-sensitive trapping agent from the compound containing the temperature-sensitive trapping agent-substrate in the presence of lyase; (s4) separating the enriched substrate; wherein the temperature-sensitive trapping agent comprises a compound as shown in a formula I, and in the formula, n = 17-90. The method is good in selectivity and high in marking efficiency, can be used for marking glycopeptide level or protein level, and has an excellent marking effect in a complex system.

Description

Technical Field

[0001] The present invention relates to the technical field of glycosylation labeling, and in particular to a method for labeling core fucosylation and / or O-GlcNAc glycosylation. Background Art

[0002] Sugar molecules have advantages such as low toxicity and good biocompatibility, which has led to increasing attention of sugar compounds and their derivatives in the field of cancer treatment. With the development of glycobiology, glycoengineering and glycomics, the application of sugars in medicine has developed rapidly.

[0003] Glycosylation is a common post-translational modification of proteins. Common protein glycosylation modifications include N-glycosylation and O-glycosylation. Among them, core fucosylation is a very important protein glycosylation that regulates the physiological and pathological processes of organisms.

[0004] Researchers are constantly developing methods to label and enrich glycosylated proteins. Currently, the most commonly used research methods include lectins, antibodies, chemical methods, metabolic labeling, and enzyme labeling. Among them, antibodies have low affinity. Lectins refer to glycoproteins or sugar-binding proteins extracted from plants, invertebrates, and higher animals that can bind to red blood cells, but their specificity is poor and has certain limitations for the study of core fucose glycosylation. Metabolic labeling specifically refers to the use of monosaccharide analogs with chemically reactive groups to culture cells, integrate the modified monosaccharide analogs into glycoconjugates in the cells through cellular metabolic pathways, and then use bioorthogonal reactions to capture and label the glycoconjugates with chemically reactive groups. Non-natural monosaccharides enter different metabolic pathways after entering the cell, resulting in the disadvantage of low selectivity. Metabolic labeling cannot be used to study glycosylation modifications in human tissue samples. Chemoenzymatic labeling involves the use of an in vitro reaction catalyzed by a glycosyltransferase or other enzyme with glycosyltransferase properties to modify target oligosaccharide determinants with non-natural sugar molecules, natural sugar molecules, or molecules directly carrying detectable or enrichable molecules, thereby labeling specific oligosaccharide structures. This method has high specificity and great potential for development and application.

[0005] Therefore, there is an urgent need in the art for a chemoenzymatic labeling method for glycosylation with good selectivity and high labeling efficiency. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for labeling core fucosylated and O-GlcNAc glycosylated polypeptides with good selectivity and high labeling efficiency.

[0007] In a first aspect of the present invention, there is provided an enrichment method comprising the following steps:

[0008] (s0) providing a premix, the premix comprising:

[0009] (a) temperature-sensitive capture agent;

[0010] (b) a sample containing a substrate to be captured, wherein the substrate to be captured is selected from the group consisting of a core fucose substrate, an O-GlcNAc substrate, or a combination thereof;

[0011] (c) a glycosylase, which is used to catalyze the ligation reaction between the temperature-sensitive capture agent and the substrate, thereby forming a "temperature-sensitive capture agent-substrate" complex;

[0012] (s1) Under enzyme catalysis conditions, the temperature-sensitive capture agent and the substrate to be captured undergo a ligation reaction, thereby forming a first mixture containing a "temperature-sensitive capture agent-substrate" complex;

[0013] (s2) adjusting the salt concentration of the first mixture to obtain a precipitate containing the "temperature-sensitive capture agent-substrate" complex, thereby obtaining a second mixture containing the precipitate;

[0014] (s3) separating the precipitate from the second mixture and dissolving it to obtain a third mixture containing a "temperature-sensitive capture agent-substrate" complex;

[0015] (s4) repeating steps (s2) to (s3) x times, wherein the third mixture containing the "temperature-sensitive capture agent-substrate" complex is used as the first mixture in step (s2), wherein x is an integer from 0 to 10;

[0016] (s5) cleaving the "temperature-sensitive capture agent-substrate" complex in the third mixture in the presence of a cleavage enzyme, thereby releasing the substrate and the temperature-sensitive capture agent from the "temperature-sensitive capture agent-substrate" complex, thereby obtaining a fourth mixture containing enriched substrate; and

[0017] (s6) separating the enriched substrate from the fourth mixture in the previous step;

[0018] in,

[0019] The temperature-sensitive trapping agent comprises a compound shown in Formula I,

[0020]

[0021] Wherein, n=17~90.

[0022] In another preferred embodiment, in step (s4), x=3-10, preferably 3-8, more preferably 3-5.

[0023] In another preferred embodiment, the step (s2) further comprises heating, and the heating refers to heating at 45-55°C, preferably 47-53°C, more preferably 48-52°C, for example 50°C.

[0024] In another preferred embodiment, the heating time is 4 to 8 minutes, preferably 4 to 7 minutes, more preferably 4 to 6 minutes, for example 5 minutes.

[0025] In another preferred embodiment, in step (s5), the method further comprises: separating the enriched substrate and temperature-sensitive capture agent from the fourth mixture.

[0026] In another preferred embodiment, the substrate is selected from the group consisting of core fucose polypeptide, O-GlcNAc polypeptide, or a combination thereof.

[0027] In another preferred embodiment, the substrate includes a core fucose polypeptide and an O-GlcNAc polypeptide.

[0028] In another preferred embodiment, the glycosylase is selected from the following group:

[0029] (c1) a first glycosylase that catalyzes the ligation reaction between the temperature-sensitive capture agent and the core fucose on the substrate;

[0030] (c2) a second glycosylase that catalyzes the ligation reaction between the temperature-sensitive capture agent and the O-GlcNAc on the substrate.

[0031] In another preferred example, the mass ratio of the first glycosylase to the second glycosylase is 20:1 to 1:20.

[0032] In another preferred embodiment, the first glycosylase is selected from the following group: EndoF3-D165A, EndoF2-D124A, or a combination thereof.

[0033] In another preferred embodiment, the second glycosylase is selected from the group consisting of EndoCC-N180H, EndoM-N175Q, or a combination thereof.

[0034] In another preferred embodiment, the first glycosylase is a mutant of β-N-acetylglucosamine endoenzyme (ENGase).

[0035] In another preferred embodiment, the first glycosylase is derived from Elizabethkingia meningoseptica.

[0036] In another preferred embodiment, the EndoCC-N180H is derived from Coprinopsis cinerea.

[0037] In another preferred embodiment, the EndoM-N175Q is derived from Mucor hiemalis.

[0038] In another preferred embodiment, the lytic enzyme is selected from the following group:

[0039] (d1) a first lyase, wherein the first lyase comprises a lyase selective for core fucose;

[0040] (d2) a second lyase, wherein the second lyase includes a lyase selective for O-GlcNAc.

[0041] In another preferred embodiment, the first lytic enzyme is selected from the following group: EndoF3, EndoF2, or a combination thereof.

[0042] In another preferred embodiment, the second lytic enzyme is selected from the group consisting of EndoCC, EndoM, or a combination thereof.

[0043] In another preferred embodiment, the first lysing enzyme is β-N-acetylglucosamine endoenzyme (ENGase).

[0044] In another preferred embodiment, the first lytic enzyme is derived from Elizabethkingia meningoseptica.

[0045] In another preferred embodiment, the EndoCC is derived from Coprinopsis cinerea.

[0046] In another preferred embodiment, the EndoM is derived from Mucor hiemalis.

[0047] In another preferred embodiment, n in the temperature-sensitive capture agent is 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 35, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, 85, 87 or 90.

[0048] In another preferred embodiment, in step (s0), the concentration of glycosylase in the premixture is 0.05-1 mg / mL.

[0049] In another preferred embodiment, in step (s0), the concentration of the glycosylase in the premix is ​​0.05-0.8 mg / mL, more preferably 0.1-0.3 mg / mL, such as 0.2 mg / mL.

[0050] In another preferred embodiment, in step (s2), the salt concentration is 0.5-1.5M.

[0051] In another preferred embodiment, the salt concentration is 0.8-2M, preferably 0.9-1.1M, for example 1M.

[0052] In a second aspect, the present invention provides a kit comprising:

[0053] (f1) temperature-sensitive capture agent;

[0054] (f2) glycosylase;

[0055] (f3) lyase; and

[0056] (f4) buffer,

[0057] in,

[0058] The temperature-sensitive trapping agent comprises a compound shown in Formula I,

[0059]

[0060] Wherein, n=17~90.

[0061] In another preferred embodiment, the buffer comprises PBS buffer.

[0062] In another preferred embodiment, n in the temperature-sensitive capture agent is 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 35, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, 85, 87 or 90.

[0063] In another preferred embodiment, the glycosylase is selected from the following group:

[0064] (c1) a first glycosylase that catalyzes the ligation reaction between the temperature-sensitive capture agent and the core fucose on the substrate;

[0065] (c2) a second glycosylase that catalyzes the ligation reaction between the temperature-sensitive capture agent and the O-GlcNAc on the substrate.

[0066] In another preferred example, the mass ratio of the first glycosylase to the second glycosylase is 20:1 to 1:20.

[0067] In another preferred embodiment, the first glycosylase is selected from the following group: EndoF3-D165A, EndoF2-D124A, or a combination thereof.

[0068] In another preferred embodiment, the second glycosylase is selected from the group consisting of EndoCC-N180H, EndoM-N175Q, or a combination thereof.

[0069] In another preferred embodiment, the first glycosylase is a mutant of β-N-acetylglucosamine endoenzyme (ENGase).

[0070] In another preferred embodiment, the first glycosylase is derived from Elizabethkingia meningoseptica.

[0071] In another preferred embodiment, the EndoCC-N180H is derived from Coprinopsis cinerea.

[0072] In another preferred embodiment, the EndoM-N175Q is derived from Mucor hiemalis.

[0073] In another preferred embodiment, the lytic enzyme is selected from the following group:

[0074] (d1) a first lyase, wherein the first lyase comprises a lyase selective for core fucose;

[0075] (d2) a second lyase, wherein the second lyase includes a lyase selective for O-GlcNAc.

[0076] In another preferred embodiment, the first lytic enzyme is selected from the following group: EndoF3, EndoF2, or a combination thereof.

[0077] In another preferred embodiment, the second lytic enzyme is selected from the group consisting of EndoCC, EndoM, or a combination thereof.

[0078] In another preferred embodiment, the first lysing enzyme is β-N-acetylglucosamine endoenzyme (ENGase).

[0079] In another preferred embodiment, the first lytic enzyme is derived from Elizabethkingia meningoseptica.

[0080] In another preferred embodiment, the EndoCC is derived from Coprinopsis cinerea.

[0081] In another preferred embodiment, the EndoM is derived from Mucor hiemalis.

[0082] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The H NMR spectrum of intermediate d (n=87, Mn 10000) is shown ( 1 1H NMR).

[0084] Figure 2 The H NMR spectrum of the thermosensitive capture agent I (n=87, Mn 10000) is shown ( 1 1H NMR).

[0085] Figure 3 Schematic diagram of the core fucosylated peptide labeling, enrichment, release, and proteomic study strategy is shown.

[0086] In the glycosylated peptide, ■ represents N-acetylglucosamine, and ▼ represents fucose. DETAILED DESCRIPTION

[0087] After extensive and in-depth research, numerous experiments and screening, the inventors unexpectedly discovered for the first time a high molecular weight temperature-sensitive capture agent, which can efficiently capture polypeptides with specific glycosylation under the action of specific enzymes, and release the captured probes, i.e., release the glycosylated polypeptides, by phase change or binding separation.

[0088] The present invention also provides a chemoenzymatic labeling method for glycosylation based on thermosensitive materials. This method has the advantages of good selectivity and high labeling efficiency. It can be used to study core fucosylation and O-GlcNAc glycosylation at the glycopeptide level, protein level, and in complex samples, and is beneficial for the study of core fucosylation and O-GlcNAc glycosylation sites. The present invention was completed on this basis.

[0089] the term

[0090] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0091] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., is understood to include the stated elements or components but does not exclude other elements or components.

[0092] As used herein, the terms "core fucosylated polypeptide," "core fucosylated protein," "core fucosylated peptide," and "core fucosylated peptide" are used interchangeably to refer to a glycosylated polypeptide with a core fucose modification.

[0093] As used herein, the terms "O-GlcNAc polypeptide," "O-GlcNAc protein," "O-GlcNAc peptide," and "O-GlcNAc glycosylated peptide" are used interchangeably to refer to a glycosylated polypeptide having an O-GlcNAc modification.

[0094] As used herein, the terms "temperature-sensitive capture agent" and "temperature-sensitive probe" are used interchangeably and both refer to active ingredients comprising the compound represented by Formula I.

[0095]

[0096] Core fucose modification

[0097] Core-fucosylation refers to the modification of fucose by linking it to the innermost GlcNAc residue of N-glycans via an α-1,6 glycosidic bond. In the human body, core-fucosylation is formed by the transfer of guanosine diphosphate (GDP)-fucose (Fuc) catalyzed by core-fucosyltransferase 8 (FUT8), and is primarily concentrated in the Golgi apparatus. As one of the most important N-glycosylation modifications, core-fucosylation has garnered extensive attention from researchers.

[0098] Compared to normal tissue, core fucose is upregulated to a certain extent in tumor tissue. Certain core-fucosylated proteins can serve as reliable cancer biomarkers. For example, alpha-fetoprotein (AFP) is an FDA-approved biomarker for hepatocellular carcinoma (HCC), and elevated core-fucosylated AFP is a specific marker for HCC. Studies have also shown that core fucose can regulate various cell surface growth factors, such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and transforming growth factor-β receptor (TGF-βR), thereby promoting tumor growth, invasion, and metastasis. Core fucose glycosylation plays an important role in regulating the immune system. The presence of core fucosylation in the Fc region of IgG inhibits antibody-dependent cell-mediated cytotoxicity (ADCC). Studies have shown that plasma from critically ill patients with novel coronavirus disease (COVID-19) contains high concentrations of core-fucosylated immunoglobulins, which amplify proinflammatory cytokine release and acute phase responses. This suggests that core fucose plays a key role in the immune response to COVID-19 and other membrane viruses.

[0099] O-GlcNAcylation

[0100] O-GlcNAc glycosylation is one of the most common glycosylation modifications. It refers to the glycosylation modification formed by the attachment of N-acetylglucose (GlcNAc) to serine or threonine residues on proteins via a glycosidic bond. O-GlcNAc glycosylation has the following characteristics: O-GlcNAc only has a monosaccharide structure and is not extended or modified into more complex glycan structures; the majority of O-GlcNAc glycosylation discovered to date is found in the cytoplasm and nucleus; and it is a highly dynamic protein modification. O-GlcNAc glycosylation participates in numerous important biological processes. O-GlcNAc modifies four histones, competing with or promoting phosphorylation or ubiquitination, thereby influencing transcription and epigenetic inheritance.

[0101] O-GlcNAcylation and phosphorylation interact with each other. On the one hand, O-GlcNAcylation sites, serine and threonine, are potential phosphorylation sites. Furthermore, O-GlcNAcylation can interact with other post-translational modifications, such as acetylation and ubiquitination. Changes in cellular O-GlcNAcylation levels are a response to a variety of physiological and pathological stimuli, and O-GlcNAcylation plays an important role in both normal physiology and disease pathology. O-GlcNAcylation is associated with metabolic diseases. Specific loss of OGT in pancreatic β cells leads to severe hyperglycemia and hypoinsulinemia. O-GlcNAcylation is also involved in neurological function and disease. Furthermore, O-GlcNAcylation is associated with cancer. Increased O-GlcNAcylation generally increases protein activation, either directly or indirectly, leading to increased gene transcription, playing a crucial role in biological physiological activities.

[0102] Methods for labeling core fucosylation and O-GlcNAc glycosylation

[0103] The present invention provides a method for labeling core fucosylation and O-GlcNAc glycosylation. Compared with existing technologies, the present method has excellent selectivity and high labeling efficiency. The present invention uses a temperature-sensitive capture agent to specifically recognize and label specific sites of core fucosylated peptides and O-GlcNAc glycosylated peptides, achieving reversible labeling of core fucosylation and O-GlcNAc glycosylation.

[0104] Typically, the method of the present invention for labeling core fucosylation and O-GlcNAc glycosylation comprises:

[0105] M1. Marking

[0106] A temperature-sensitive capture agent and an ENGase mutant selective for the core fucose substrate and the O-GlcNAc substrate are added to the core fucose and O-GlcNAc glycosylated peptide segments, and the reaction is carried out at 25-37° C. for 1-5 hours to obtain a first reaction mixture.

[0107] In another preferred embodiment, the ENGase mutant selective for core fucose substrate is EndoF3-D165A or EndoF2-D124A.

[0108] In another preferred embodiment, the ENGase mutant selective for core fucose substrate is EndoF3-D165A.

[0109] In another preferred embodiment, the ENGase mutant selective for O-GlcNAc substrate is EndoCC-N180H, EndoM-N175Q, or a combination thereof.

[0110] In another preferred embodiment, the ENGase mutant selective for O-GlcNAc substrate is EndoCC-N180H.

[0111] In another preferred embodiment, the final concentration of the ENGase mutant selective for the core fucose substrate is 0.05-1 mg / mL, preferably 0.05-0.5 mg / mL, more preferably 0.1-0.3 mg / mL, for example 0.2 mg / mL.

[0112] In another preferred embodiment, the EndoF3-D165A and EndoF2-D124A are mutants of β-N-acetylglucosamine endoenzyme (ENGase).

[0113] In another preferred embodiment, the EndoF3-D165A and EndoF2-D124A are derived from Elizabethkingiameningoseptica.

[0114] In another preferred embodiment, the EndoCC-N180H is derived from Coprinopsis cinerea.

[0115] In another preferred embodiment, the EndoM-N175Q is derived from Mucor hiemalis.

[0116] M2. Enrichment

[0117] Add sodium chloride to the first reaction mixture in M1, heat at 40-60°C for 3-10 minutes, centrifuge, and collect the precipitate. Repeat the above steps 2-3 times to obtain a solid precipitate.

[0118] In another preferred embodiment, the reaction is carried out at 45-55°C, for example 50°C.

[0119] In another preferred embodiment, the heating time is 4 to 8 min, preferably 4 to 7 min, more preferably 4 to 6 min, for example 5 min.

[0120] In another preferred embodiment, the final concentration of sodium chloride added to the first solvent is 0.8-2M, preferably 0.9-1.1M, for example 1M.

[0121] M3. Release

[0122] Dissolve the solid precipitate obtained in M2 with PBS and add ENGase, which is selective for core fucose and O-GlcNAc. Incubate at 20-30°C for 3-15 hours. Add sodium chloride and heat at 40-60°C for 3-10 minutes. Collect the supernatant to obtain a peptide labeled for core fucose and O-GlcNAc glycosylation.

[0123] In another preferred embodiment, the ENGase selective for core fucose is EndoF3 or EndoF2.

[0124] In another preferred embodiment, the ENGase selective for core fucose is EndoF3.

[0125] In another preferred embodiment, the EndoF3 and EndoF2 are β-N-acetylglucosamine endoenase (ENGase).

[0126] In another preferred embodiment, the EndoF3 and EndoF2 are derived from Elizabethkingia meningoseptica.

[0127] In another preferred embodiment, the ENGase selective for O-GlcNAc is EndoCC or EndoM.

[0128] In another preferred embodiment, the ENGase selective for O-GlcNAc is EndoCC.

[0129] In another preferred embodiment, the EndoCC is derived from Coprinopsis cinerea.

[0130] In another preferred embodiment, the EndoM is derived from Mucor hiemalis.

[0131] The main advantages of the present invention include:

[0132] (1) The present invention provides a chemical enzyme labeling method using a temperature-sensitive capture agent, which can simultaneously achieve the labeling of two glycosylation residues with excellent selectivity and labeling efficiency;

[0133] (2) The method of the present invention can be used for core fucosylation and O-GlcNAc glycosylation labeling at the glycopeptide level or protein level;

[0134] (3) The method of the present invention has excellent labeling effect in complex systems;

[0135] (4) The method of the present invention can be used to study core fucosylation and O-GlcNAc glycosylation at the glycopeptide level, protein level, or complex samples.

[0136] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight. Test reagents are commercially available.

[0137] Material

[0138] Egg yolk powder was purchased from Anhui Rongda Food Company.

[0139] Propargylamine was purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0140] Tert-Butyloxycarbonyl-11-aminoundecanoic acid was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0141] Carboxylic acid-terminated poly (N-isopropylacrylamide) (Mn 10000, product number: 724459) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0142] Tert-butyl (3-aminopropyl) carbamate (CAS No. 75178-96-0) was purchased from Shaoyuan Technology (Shanghai) Co., Ltd.

[0143] O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, CAS No.: 148893-10-1) was purchased from Accela.

[0144] N,N-Diisopropylethylamine (DIPEA; CAS No.: 7087-68-5) was purchased from Maclean.

[0145] Methanol (CAS No.: 67-56-1) and dichloromethane (CAS No.: 75-09-2) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0146] Triethylamine (Et3N, CAS No.: 121-44-8), N,N-dimethylformamide (CAS No.: 68-12-2), and trifluoroacetic acid (CAS No.: 76-05-1) were purchased from J&K.

[0147] The core fucose standard glycopeptide P1 and the O-GlcNAc standard glycopeptide P2 were synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0148] Sodium lauryl sulfate was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0149] The sugar chain probe (compound of formula I) was synthesized in the laboratory.

[0150] 9-azidosialic acid was synthesized according to the method reported in a known literature (ACS Central Science 2020, 6(3), 382-389.).

[0151] Example 1 Enzyme expression and purification

[0152] Enzymes NanA (sialidase from Streptococcus pneumoniae, Uniprot ID: P62576), Pd2,6ST (sialyltransferase from Photobacterium damselae, Uniprot ID: O66375), NmCSS (N-acylneuraminic acid cytidyltransferase from Neisseria meningitidis, Uniprot ID: P0A0Z8), EndoF3 (Uniprot ID: P36913), EndoF3-D165A (Uniprot ID: P36913, amino acid 165 is mutated from aspartic acid to alanine), EndoF2 (Uniprot ID: P36912), EndoF2-D124A (Uniprot ID: P36912, amino acid 124 is mutated from aspartic acid to alanine after removal of the signal peptide (1-45 amino acids), EndoCC ... Expression and purification of EndoCC-N180H (Uniprot ID: A8P7P2, amino acid 180 was mutated from asparagine to histidine), EndoM (Uniprot ID: Q9C1S6), and EndoM-N175Q (Uniprot ID: Q9C1S6, amino acid 175 was mutated from asparagine to glutamine).

[0153] The NanA, Pd2,6ST, NmCSS, EndoF3, EndoF3-D165A, EndoF2, EndoF2-D124A, EndoM, and EndoM-N175Q genes were synthesized by Beijing Liuhe BGI Genomics Co., Ltd. and ligated into the pET28a vector (kanamycin resistance). Competent cells were transformed, positive clones were selected, and cultured. Escherichia coli expression strain BL21(DE3) (Thermo Fisher Scientific, EC0114) containing the above-mentioned vectors was cultured at 37°C. When the OD600 value was approximately 0.7, 0.1 mM isopropyl-β-D-thiogalactose (IPTG) and 10% glycerol were added and protein expression was induced at 16°C for 30 h. The cells were harvested by centrifugation at 4°C. Bacteria were resuspended in PBS and disrupted by sonication. The supernatant was centrifuged and incubated with gel affinity resin (Ni-NTA agarose, Thermo Fisher Scientific, R90101) at 4°C for 1 hour. The supernatant was washed with 10 mM imidazole in PBS and eluted with 300 mM imidazole in PBS. The imidazole was removed using ultrafiltration tubes (Millipore, UFC9010). 20% glycerol was added to the protein solution in PBS, and the aliquots were frozen. Protein purity and concentration were determined by SDS-PAGE.

[0154] Example 2 Synthesis of Thermosensitive Capture Agent Precursor Molecule

[0155] (1) Synthesis of octasaccharide h

[0156]

[0157] According to the method reported in the known literature (Nat. Protoc. 2017, 12, 1702–1721.), sialylglycopeptide (SGP, compound g) was extracted from egg yolk powder. SGP was dissolved in PBS solution (pH 6.0), and enzymes EndoM and NanA were added. The reaction was carried out at 30°C for 4 h. The reaction was monitored by thin layer chromatography (TLC, isopropanol:ammonia:water = 7:3:2, v:v:v) and the octasaccharide h was separated and purified by polyacrylamide gel P-2 (Bio-Rad, #1504118).

[0158] (2) Synthesis of sugar chain precursor (compound e)

[0159]

[0160] NmCSS (0.2 mg / mL) and Pd2,6ST (0.1 mg / mL) purified in Example 1 were added to a PBS solution (100 mM, pH 7.5, 10 mM MgCl2, 10 mL) containing octasaccharide h (200 mg, 0.14 mmol), cytidine triphosphate disodium (142 mg, 0.3 mmol) and 9-azidosialic acid (67 mg, 0.2 mmol). The reaction was incubated at 37°C and the reaction progress was monitored by TLC (developing solvent: isopropanol:ammonia:water = 7:3:2, v:v:v). After 4 h, the reaction was basically completed, and the supernatant was collected by centrifugation and separated and purified on polyacrylamide gel P-2 to obtain the target sugar chain probe precursor (compound e).

[0161] Example 3 Synthesis of Temperature-Sensitive Capture Agent

[0162] The schematic diagram of the synthesis process of the temperature-sensitive capture agent is shown below.

[0163]

[0164] (1) Synthesis of intermediate b

[0165] To DCM (5 mL) were added propargylamine (55 mg, 1 mmol), tert-butyloxycarbonyl-11-aminoundecanoic acid (348 mg, 1 mmol), HATU (760 mg, 2 mmol), and DIPEA (740 μL, 4 mmol). The mixture was stirred under nitrogen for 4 h. The reaction solution was evaporated to dryness using a rotary evaporator, purified by silica gel column, and freeze-dried to obtain intermediate b.

[0166] (2) Synthesis of intermediate c

[0167] The crude product b was placed in a round-bottom flask, and equal volumes of dichloromethane (DCM) and trifluoroacetic acid (TFA) were added. The reaction was carried out on a magnetic stirrer under nitrogen protection at room temperature for 1 hour. The reaction solution was evaporated to dryness using a rotary evaporator and then dissolved in a small amount of ether. After ultrasonication to precipitate crystals, the intermediate c was obtained by filtration.

[0168] (3) Synthesis of intermediate d

[0169] To DCM (10 mL) were added intermediate c (71.4 mg, 0.03 mmol), carboxylic acid-terminated poly(N-isopropylacrylamide) (PNIPAM) (1 g, 0.01 mmol), HATU (190 mg, 0.05 mmol), and DIPEA (163 μL, 0.01 mmol), and the mixture was stirred under nitrogen for 8 h. The reaction solution was diluted with 1 volume (10 mL) of water, cooled, and centrifuged to obtain the supernatant, which was then purified by dialysis (MWCO 12000). After dialysis, the solution was precipitated, the supernatant was concentrated, and lyophilized. The crude product was purified by Sephadex G50 column (water) and lyophilized to obtain the crude product.

[0170] The dried sample was dissolved in 10 mL of water, and compound e (62 mg, 0.03 mmol), copper sulfate (0.006 mmol), sodium ascorbate (0.03 mmol), and THTPA (0.012 mmol) were added. The reaction was allowed to react for about 30 min. The reaction was monitored with isopropanol: ammonia: water = 7:3:2. When the reaction no longer proceeded, it was dialyzed (MWCO 12000) for purification. The crude product was purified with Sephadex G50 (water) and lyophilized to obtain intermediate d.

[0171] (4) Synthesis of thermosensitive capture agents

[0172] Intermediate d (15 mg) was dissolved in 188 μL of deionized water, and 8 μL of N,N-dimethylformamide (DMF) and 4 μL of triethylamine (Et3N) were added. The mixture was ice-bathed for 30 min, and 4.0 M aqueous sodium chloride solution (100 μL) was added. After heating for 5 min, a precipitate appeared. The supernatant was discarded, and the precipitate was redissolved in water on ice to obtain the thermosensitive capture agent.

[0173] The H NMR spectra of intermediate d and thermosensitive capture agent (n=87, Mn 10000) are shown in Figure 2. Figure 1 and Figure 2 shown.

[0174] Example 4 Reversible Labeling of Standard Peptides

[0175] Schematic diagram of reversible labeling, enrichment and release Figure 3 shown.

[0176] To a PBS buffer (200 mM, pH = 7.5) containing 0.5 mM standard peptides (core fucose standard glycopeptide P1 and O-GlcNAc standard glycopeptide P2, 0.5 mM each), 45 mg of the temperature-sensitive probe (compound 1, 15 mg each time) was added three times in PBS buffer (200 mM, pH = 7.0, a total volume of 600 μL) and mixed. The enzyme was added to a final concentration of 0.2 mg / mL (EndoF3-D165A for P1 and EndoCC-N180H for P2) and reacted at room temperature for 3 hours. After the reaction was completed, 4.0 M sodium chloride aqueous solution (200 μL) was added. The mixture was heated at 50 ° C for 5 minutes. Centrifuged at 10,000 × g for 5 minutes, the supernatant was analyzed by HPLC to obtain the reduction in the core fucose standard glycopeptide P1. The precipitate was dissolved in water (600 μL) in an ice bath. Then, add 4.0M sodium chloride aqueous solution (200μL). Heat at 40℃ for 5min. Centrifuge at 10000×g for 5min and collect the precipitate. Repeat the dissolution of the precipitate three times to wash away the unlabeled peptides. The third supernatant is retained for HPLC analysis to ensure that the impurities are washed away. Dissolve the third precipitate in PBS buffer (100mM, pH=7.0, 600μL) in an ice bath. Then add endoglycosidase (P1 is EndoF3, P2 is EndoCC, final concentration is 0.1mg / mL). React at room temperature for 12h. After the reaction is completed, add 4.0M sodium chloride aqueous solution (200μL). Heat the mixture at 50℃ for 5min. Centrifuge at 10000×g for 5min, and analyze the supernatant by HPLC to obtain the amount of released standard glycopeptides. The enrichment and release processes were monitored by reverse phase HPLC (Table 1) at 214 nm using a linear gradient elution (elution conditions are as follows: A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile) by reverse phase HPLC (Shimadzu, C18 column: Spherisorb, 5 μm, 250×4.6 mm).

[0177] Table 1

[0178]

[0179] The experimental results show that this method can efficiently label, enrich, and release core-fucosylated standard peptides. Core-fucosylated peptides can be effectively enriched for further research.

[0180] Example 5 Reversible labeling of standard peptides in complex systems

[0181] (1) BSA peptide acquisition

[0182] Weigh 100 mg of BSA solid and dissolve it in 10 mL of 100 mM NH₄HCO₃ buffer. Add trypsin. Incubate at 37°C for 24 hours, adding trypsin once. After digestion, desalt the BSA peptide using C18 (Sep-Pak tC18) solid-phase extraction and lyophilize. Dissolve the lyophilized solid in ddH₂O, determine the concentration using a BCA protein assay kit, and store at -80°C until use.

[0183] The C18 solid-phase extraction process described above: 1 mL of methanol was added to a Sep-Pak tC18 cartridge for activation. Once no liquid remained above the cartridge filler, 1 mL of 70% acetonitrile (containing 0.1% TFA) in water was added for further activation. After activation, 1 mL of 0.1% TFA was added for equilibration. Before loading the peptide, the sample was acidified with 0.1% TFA and centrifuged to remove precipitates. The sample volume was kept around 1 mL. The sample was washed twice with 1 mL of 0.1% TFA in water. Elution was then performed with 70% acetonitrile (containing 0.1% TFA) in water. The resulting eluate was lyophilized.

[0184] (2) Reversible labeling of standard glycopeptides in complex systems

[0185] The standard peptides (core fucose standard glycopeptide P1 (SEQ ID NO: 1 (EEQY (Fucα1, 6GlcNAcβ) NSTYR) or O-GlcNAc standard glycopeptide P2 (SEQ ID NO:2 (TAPT (GlcNAcβ) STIAPG), 20 μg each) and digested BSA peptide (10 mg) were mixed in PBS buffer (200 mM, pH = 7.0) at a ratio of 1:500 (w / w). 45 mg of the temperature-sensitive probe (compound 1, 15 mg each time, 1 h apart) was added to the resulting peptide mixture in three additions for a total of 3 h. After the reaction, 4.0 M sodium chloride aqueous solution (200 μL) was added. The mixture was heated at 40 ° C for 5 min. Centrifuged at 10,000 × g for 5 min, the solid was collected and redissolved in water (600 μL). Then, 4.0 M sodium chloride aqueous solution (200 μL) was added. Heat at 50 ° C for 5 min. Centrifuged at 10,000 × g for 5 min, and the precipitate was collected. Repeat the dissolution of the precipitate ten times to wash away the unlabeled peptides.

[0186] The washed solid was dissolved in PBS buffer (50 mM, pH = 7.0, 600 μL). Then, endoglycosidase EndoF3 (final concentration of 0.1 mg / mL) was added. The mixture was reacted at room temperature for 12 h. Then, 4.0 M sodium chloride aqueous solution (200 μL) was added. The temperature of the mixture was raised to 40°C to precipitate the PNIPAM compound. The supernatant was collected by centrifugation (10,000 × g). The release of the standard peptide in the supernatant was subjected to C18 solid phase extraction desalting (Sep-Pak tC18 cartridge) and analyzed by LC-MS / MS.

[0187] Example 6 Reversible labeling of target peptides in cell peptides

[0188] (1) Acquisition of tumor cell peptides

[0189] Human cervical cancer cells Hela were cultured in DMEM medium containing 10% FBS, 100 U / ml penicillin, and 100 U / ml streptomycin at 37°C and 5% CO2. 10 mM TMG (a small molecule compound that inhibits O-GlcNAc glycosylation hydrolysis) was added 24 hours before cell harvest.

[0190] Cells were harvested and lysed with 4% SDS (1M Tris) at 4°C for 5 minutes. The mixture was centrifuged at 4°C, and the supernatant was collected. 20 mM dithiothreitol (DTT) was added to the supernatant and incubated at 37°C for 2 hours. The mixture was then cooled to room temperature, and 40 mM iodoacetamide (IAA) was added to the reaction mixture. The reaction mixture was incubated in the dark for 40 minutes at room temperature. Protein precipitation was then performed (methanol:chloroform:water volume ratio = 4:1:4). 8 M urea was added to the protein precipitate to dissolve the protein, and the protein solution was diluted with 100 mM NH4HCO3 buffer to a final urea concentration of 1 M. Trypsin (Promega enzyme:substrate mass ratio 1:100) was added and digested at 37°C for 34 hours. The resulting peptide solution was desalted using C18 solid-phase extraction (Sep-Pak tC18 cartridge) and lyophilized. PBS buffer (100 mM, pH = 7.5) was added to dissolve the lyophilized peptides, and the peptide concentration was detected by Nanodrop. The final concentration of the peptides diluted in PBS solution was 5 mg / mL. EndoF3 (0.1 mg / mL) was then added and reacted at 37°C for 2 hours to expose the core fucose sites. PNGase F (0.05 mg / mL) was then added and reacted at 37°C for 2 hours to expose the core fucose sites. The deglycosylated peptides were then desalted by C18 solid-phase extraction (Sep-Pak tC18 cartridge) and lyophilized. The purified deglycosylated peptide solid was dissolved in water, aliquoted, and stored at -80°C. The concentration of the deglycosylated peptide was determined by Nanodrop.

[0191] (2) Reversible labeling of glycosylation of tumor cell peptides

[0192] 45 mg of the thermosensitive probe (compound 1, 15 mg each time, 1 h apart) was added to a PBS buffer solution containing 3 mg of the deglycosylated peptide obtained in (1) above (1) three times, and EndoF3-D165A (final concentration of 0.2 mg / mL) was added at the same time and reacted at 25°C for 3 h. After the reaction, 4.0 M sodium chloride aqueous solution (200 μL) was added. The mixture was heated at 50°C for 5 min. Centrifuged at 10,000 × g for 5 min, the solid was collected and redissolved in water (600 μL). Then, 4.0 M sodium chloride aqueous solution (200 μL) was added. Heat at 40°C for 5 min. Centrifuged at 10,000 × g for 5 min, and the precipitate was collected. Repeat the dissolution of the precipitate ten times to wash away the unlabeled peptides.

[0193] The washed solid was dissolved in PBS buffer (50 mM, pH = 7.0, 600 μL). Endoglycosidase EndoF3 (final concentration 0.1 mg / mL) was then added. The mixture was reacted at 25°C for 12 h. A 4.0 M sodium chloride aqueous solution (200 μL) was then added. The temperature of the mixture was raised to 40°C to precipitate the PNIPAM compound. The mixture was centrifuged at 10,000 × g and the supernatant was collected. The supernatant was desalted by C18 solid-phase extraction (Sep-Pak tC18 cartridge).

[0194] After reversibly labeling, peptide samples from tumor cells were analyzed using LC-MS / MS and database search was performed. Ultimately, 883 core fucose sites and 519 O-GlcNAc glycosylation sites were identified.

[0195] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An enrichment method, characterized in that, The method comprises the following steps: (s0) providing a premix, the premix comprising: (a) temperature-sensitive capture agent; (b) a sample containing a substrate to be captured, wherein the substrate to be captured is selected from the group consisting of a core fucose substrate, an O-GlcNAc substrate, or a combination thereof; (c) a glycosylase, which is used to catalyze the ligation reaction between the temperature-sensitive capture agent and the substrate, thereby forming a "temperature-sensitive capture agent-substrate" complex; (s1) Under enzyme catalysis conditions, the temperature-sensitive capture agent and the substrate to be captured undergo a ligation reaction, thereby forming a first mixture containing a "temperature-sensitive capture agent-substrate" complex; (s2) adjusting the salt concentration of the first mixture to obtain a precipitate containing the temperature-sensitive capture agent-substrate complex, thereby obtaining a second mixture containing the precipitate; (s3) separating the precipitate from the second mixture and dissolving it to obtain a third mixture containing a "temperature-sensitive capture agent-substrate" complex; (s4) repeating steps (s2) to (s3) x times, wherein the third mixture containing the "temperature-sensitive capture agent-substrate" complex is used as the first mixture in step (s2), wherein x is an integer from 0 to 10; (s5) cleaving the "temperature-sensitive capture agent-substrate" complex in the third mixture in the presence of a cleavage enzyme, thereby releasing the substrate and the temperature-sensitive capture agent from the "temperature-sensitive capture agent-substrate" complex, thereby obtaining a fourth mixture containing enriched substrate; and (s6) separating the enriched substrate from the fourth mixture in the previous step; in, The temperature-sensitive trapping agent comprises a compound shown in Formula I, Wherein, n=17~90.

2. The method according to claim 1, wherein The substrate is selected from the group consisting of a core fucose polypeptide, an O-GlcNAc polypeptide, or a combination thereof.

3. The method according to claim 1, wherein The glycosylase is selected from the group consisting of: (c1) a first glycosylase that catalyzes the ligation reaction between the temperature-sensitive capture agent and the core fucose on the substrate; (c2) a second glycosylase that catalyzes the ligation reaction between the temperature-sensitive capture agent and the O-GlcNAc on the substrate.

4. The method according to claim 1, wherein The lytic enzyme is selected from the group consisting of: (d1) a first lyase, wherein the first lyase comprises a lyase selective for core fucose; (d2) a second lyase, wherein the second lyase includes a lyase selective for O-GlcNAc.

5. The method according to claim 1, wherein In the temperature-sensitive capture agent, n=17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 35, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, 85, 87 or 90.

6. The method according to claim 1, wherein In step (s0), the concentration of glycosylase in the premixture is 0.05-1 mg / mL.

7. The method according to claim 1, wherein In step (s2), the salt concentration is 0.5-1.5M.

8. A kit, characterized in that include: (f1) temperature-sensitive capture agent; (f2) glycosylase; (f3) Lyase; and (f4) buffer, in, The temperature-sensitive trapping agent comprises a compound shown in Formula I, Wherein, n=17~90.

9. The kit according to claim 8, wherein The glycosylase is selected from the group consisting of: (c1) a first glycosylase that catalyzes the ligation reaction between the temperature-sensitive capture agent and the core fucose on the substrate; (c2) a second glycosylase that catalyzes the ligation reaction between the temperature-sensitive capture agent and the O-GlcNAc on the substrate.

10. The kit according to claim 8, wherein The lytic enzyme is selected from the group consisting of: (d1) a first lyase, wherein the first lyase comprises a lyase selective for core fucose; (d2) a second lyase, wherein the second lyase includes a lyase selective for O-GlcNAc.

Images