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A method for enriching n-glycopeptides or n-sugar chains

A sugar chain and enrichment technology, applied in the field of enriching N-glycopeptides or N-glycosides, can solve the problems of complex operation steps, time-consuming and high technical requirements

Active Publication Date: 2021-07-06
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the operation steps are complex, technically demanding, and time-consuming. In large-scale glycoproteome and glycogen analysis, it is necessary to have an effective, easy-to-operate, and rapid method to enrich glycopeptides and glycans before MS analysis. chain

Method used

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  • A method for enriching n-glycopeptides or n-sugar chains
  • A method for enriching n-glycopeptides or n-sugar chains
  • A method for enriching n-glycopeptides or n-sugar chains

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Experimental program
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Effect test

Embodiment 1

[0044] The enrichment of embodiment 1N-glycopeptide

[0045] Bacterial cellulose pieces were cut into 1 cm × 1 cm pieces to obtain BC pieces, which were washed 3 times with distilled water, and then, BC pieces were freeze-dried and stored at 4 °C for further use.

[0046] Proteins were diluted in 50 mM ammonium bicarbonate (ABC) buffer at a concentration of 2 μg / μL and denatured in a 100° C. water bath for 5 min. Proteins were then reduced with 10 mM DTT for 1 h at 37°C, followed by alkylation with 25 mM IAA for 0.5 h at 37°C. Then trypsin was added to the solution at an enzyme:protein ratio of 1:50 (w / w), incubated overnight at 37°C, and then boiled at 100°C for 5min to terminate the enzymatic hydrolysis reaction. Digested peptides were dried by vacuum centrifugation and stored at -20°C for later use.

[0047] To obtain a mixture of sugar chains from the peptide, the lyophilized peptide from the last step was dissolved in 50 mM ABC, and the peptide N- Glycosidase F release...

Embodiment 2

[0049] Example 2 Enrichment of N-sugar chains

[0050] Bacterial cellulose pieces were cut into 1 cm × 1 cm pieces to obtain BC pieces, which were washed 3 times with distilled water, and then, BC pieces were freeze-dried and stored at 4 °C for further use.

[0051] Proteins were diluted in 50 mM ammonium bicarbonate (ABC) buffer at a concentration of 2 μg / μL and denatured in a 100° C. water bath for 5 min. Proteins were then reduced with 10 mM DTT for 1 h at 37°C, followed by alkylation with 25 mM IAA for 0.5 h at 37°C. Then trypsin was added to the solution at an enzyme:protein ratio of 1:50 (w / w), incubated overnight at 37°C, and then boiled at 100°C for 5min to terminate the enzymatic hydrolysis reaction. Digested peptides were dried by vacuum centrifugation and stored at -20°C for later use.

[0052] To obtain the glycan mixture from the peptide, the lyophilized peptide in the last step was dissolved in 50 mM ABC, and the glycan peptide was added at an enzyme:protein ra...

Embodiment 3

[0054] Example 3 Characterization of bacterial cellulose

[0055] Bacterial cellulose is composed of sugar chains with β-1,4-glycosidic bonds and has an interconnected 3D porous network structure. The morphology of bacterial cellulose was observed by SEM. Such as image 3 As shown in A and 3B, bacterial cellulose has a nanoscale fiber structure. The interconnected 3D porous network structure provides high specific surface area, strong flexibility and high tensile strength. Bacterial cellulose has a remarkable water absorption capacity. Bacterial cellulose forms solid sheets after drying, while the solution swells like a gel after absorption ( image 3 C). Significant water capacity was evaluated by mass change before and after water absorption. Bacterial cellulose can absorb 80 times its own weight in water.

[0056] The nitrogen adsorption-desorption isotherm further proved the porous structure of bacterial cellulose ( image 3 D). The hysteresis loop representing th...

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Abstract

The invention discloses a method for enriching N-glycopeptides or N-sugar chains. The method comprises pretreating bacterial cellulose, denaturing and enzymatically digesting the protein solution, and then pretreating the pretreated bacteria Cellulose is added to the treated protein solution for enrichment. The present invention identifies 36 and 159 kinds of N-glycan chains and 31 and 523 complete N-glycopeptides in IgG standard protein and human serum samples, and the whole enrichment process is very easy to operate, and the speed is super fast (only 10 minutes) , with a high specificity of up to 94%. Enrichment is not affected by salt, allowing samples to be directly enriched after enzymatic digestion without desalting.

Description

technical field [0001] The invention relates to the technical field of glycopeptide enrichment, in particular to a method for enriching N-glycopeptides or N-sugar chains. Background technique [0002] Glycosylation has long been recognized as one of the most common and important protein modification processes in living organisms and has a great influence on many biological processes. Sugar chains on proteins have a variety of functional properties, from providing the structural composition of cells to regulating their physical and chemical properties. Abnormal protein glycosylation is directly related to various diseases, such as cancer, neurodegenerative diseases, immune-related diseases, etc. Due to the importance of protein glycosylation, there has been considerable interest in developing new methods and conducting related research in the fields of glycoproteomics and glycomics. [0003] At present, many effective mass spectrometry (MS)-based protein glycosylation analy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/38C07K1/22C12P19/14C12P21/06G01N1/40G01N30/08G01N33/68
CPCB01D15/38C07K1/22C12P19/14C12P21/06G01N1/405G01N30/08G01N33/6848
Inventor 曹纬倩吴梦溪张权青杨芃原
Owner FUDAN UNIV
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