Preparation method of solid-phase enriched O-GlcNAc glycopeptide

A glycopeptide and enrichment technology, applied in the field of biomolecule preparation and analysis, can solve the problems of enrichment of O-GlcNAc glycopeptides and low site specificity

Pending Publication Date: 2022-03-08
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for preparing solid-phase enrichment of O-GlcNAc glycopeptides

Method used

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  • Preparation method of solid-phase enriched O-GlcNAc glycopeptide
  • Preparation method of solid-phase enriched O-GlcNAc glycopeptide
  • Preparation method of solid-phase enriched O-GlcNAc glycopeptide

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preparation example Construction

[0041] See figure 1 , the present application discloses a preparation method for solid-phase enrichment of O-GlcNAc glycopeptides, which comprises the following steps:

[0042] S1. Provide a polypeptide mixture and a solid phase material, the polypeptide mixture includes glycan-free polypeptides, N-glycan polypeptides, mucin-type O-glycopeptides and O-GlcNAc glycopeptides;

[0043] S21. Oxidize the polypeptide mixture and covalently bind to the solid phase material to obtain solid phase bound O-glycopeptides and O-GlcNAc glycopeptides after removing N-glycans;

[0044] or,

[0045] S22. After removing the N-glycans of the polypeptide mixture, affinity-binding the polypeptide mixture on the solid phase material to obtain solid phase bound O-glycopeptide and O-GlcNAc glycopeptide;

[0046] as well as,

[0047] S3. Further enzyme digestion or hydrolysis to obtain O-GlcNAc glycopeptide.

[0048] Specifically, in S21, the polypeptide mixture is oxidized and covalently bound to ...

Embodiment 1

[0064] Example One Covalent Bonding Preparation of Solid-Phase-Bound O-Glycopeptides

[0065] See figure 2 ,specific:

[0066] Freeze the tissue in dry ice or -80°C for 2-3 hours, and break the tissue immediately after taking it out;

[0067] Add 400-600 μl 1 times RIPA lysate to the tissue, use an ultrasonic breaker with 30-40% energy, break the sample for 30 seconds and put the sample in ice to cool, repeat this step 4-6 times until the sample solution is clear;

[0068] Take 2-4μl sample, dilute it 5-10 times, and test the protein concentration with BCA;

[0069] According to the concentration, take 800-1000μg protein and dissolve it in urea with a total volume of 400-600μl and a final concentration of 8M, shake the sample slightly to ensure that the protein is completely dissolved;

[0070] Add 80-100μl 120mM dithrethiol (DTT), and react at 37°C for 1-1.5 hours;

[0071] Then add 80-100μl 160mM iodoacetamide and react in dark room at room temperature for 1-1.5 hours; ...

Embodiment 2

[0082] Example 2 Affinity Binding Preparation of O-Glycopeptides Binding to Solid Phase

[0083] See image 3 ,specific:

[0084] Vacuum freeze-dry 800-1000 μg of C18 purified polypeptide and redissolve in 400-600 μl PBS buffer, pH 7.2-7.6;

[0085] Add 1-2 units of N-glycosidase (PNGaseF) to the sample, react at 37°C for 4-6 hours, so that the N-glycan can be removed by enzymatic digestion, and glycan-free polypeptide, mucin-type O-glycopeptide, O- GlcNAc glycopeptides and N-glycans;

[0086] Add 20-30 μl concentrated TFA (>99%, w / v) to the sample to lower the pH to below 3.0, purify with C18 to remove N-glycans, and vacuum freeze-dry the purified sample;

[0087] Purified samples were redissolved in 400-500 μl lectin WGA buffer, i.e. 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid), 0.15M NaCl, pH 7.5;

[0088] Rinse 200-300μl WGA lectin spherical resin and 400-500μl WGA buffer 2-3 times, keep the WGA resin, and remove the washing solution;

[0089] Ad...

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Abstract

The invention relates to a preparation method of solid-phase enriched O-GlcNAc glycopeptide, which comprises the following steps: S1, providing a polypeptide mixture and a solid-phase material, the polypeptide mixture comprising glycan-free polypeptide, N-glycan polypeptide, mucoprotein type O-glycopeptide and O-GlcNAc glycopeptide; s21, oxidizing the polypeptide mixture, covalently binding the oxidized polypeptide mixture to a solid-phase material, and removing N-glycan to obtain O-glycopeptide and O-GlcNAc glycopeptide which are subjected to solid-phase binding; or S22, removing N-glycan of the polypeptide mixture, and binding the polypeptide mixture to a solid-phase material through affinity to obtain O-glycopeptide and O-GlcNAc glycopeptide which are subjected to solid-phase binding; and S3, further performing enzyme digestion or hydrolysis to obtain the O-GlcNAc glycopeptide. The O-glycopeptide enriched with the O-GlcNAc glycopeptide and the site specificity of the O-glycopeptide prepared by the method are both at a relatively high level, so that the problem of low O-GlcNAc glycopeptide enriched with the O-GlcNAc glycopeptide and the site specificity of the O-glycopeptide prepared in the prior art is solved.

Description

technical field [0001] The invention relates to a preparation method for solid-phase enrichment of O-GlcNAc glycopeptides, belonging to the technical field of biomolecular preparation and analysis. Background technique [0002] O-GlcNAcylation is a nutrient and stress-responsive post-translational modification (PTM) that involves linking O-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of cytoplasmic, nuclear, and mitochondrial proteins. A pair of enzymes that add and remove O-GlcNAc, O-GlcNAc transferase (OGT) and O-GlcNAcase (O-GlcNA glycosidase, OGA), control the dynamic cycle of this PTM. O-GlcNAcylation is involved in the spatiotemporal regulation of diverse cellular processes, including transcription, epigenetic modification, and cell signaling dynamics. O-GlcNAcylation is highly dynamic and often transient, but the mechanisms underlying the temporal control of O-GlcNAcylation signaling are largely unknown. O-GlcNAcylation can be viewed as the essential "grea...

Claims

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Application Information

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IPC IPC(8): C07K1/107C12P21/06C12P21/00C07K1/04C07K1/12C07K1/14C07K1/36
CPCC07K1/1077C12P21/06C12P21/005C07K1/12C07K1/04C07K1/14C07K1/145C07K1/36
Inventor 杨霜胡文华
Owner SUZHOU UNIV
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