Method for rapid enzymolysis release, solid phase concentration and mass spectrometry of N-glycan

A sugar chain and enzymatic hydrolysis technology, which is applied in the field of glycoproteomics and glycomics analysis, can solve the problems of high price, long operation time, and low proportion of glycosylation modification, so as to improve the selectivity and binding speed of mass spectrometry analysis The effect of fast and strong affinity

Active Publication Date: 2020-05-01
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the research on N-glycans faces the following difficulties: First, although there are abundant types of N-glycosylated proteins, the proportion of glycosylation modifications is relatively low, in other words, the overall abundance of N-glycans is at a low level; Secondly, there is microscopic heterogeneity in N-glycan chains, that is, there may be N-glycan chains with different compositions on the same glycosylation site, so that the abundance of a single N-glycan chain is also at a low level, which affects the sensitivity of the analytical method. There are higher requirements; again, in the process of mass spectrometry, the N-glycan chain lacks hydrophobic groups and groups with stable charges, the ionization efficiency is low, and the signal is more likely to be easily ionized by peptides, proteins, etc. in the sample. Substance inhibition; Finally, although there are some tools such as microcolumns and micropipettes for N-glycan enrichment, they often have problems of low selectivity, high price and long operation time

Method used

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  • Method for rapid enzymolysis release, solid phase concentration and mass spectrometry of N-glycan
  • Method for rapid enzymolysis release, solid phase concentration and mass spectrometry of N-glycan
  • Method for rapid enzymolysis release, solid phase concentration and mass spectrometry of N-glycan

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Effect test

Embodiment 1

[0039] For solid-phase enrichment of samples containing free N-glycans

[0040] Weigh 3-7mg absorbent cotton material, fill it into the tip of the pipette, and place it on the centrifuge tube. Use 50-80 μL of water to activate once, centrifuge to remove the solution, the interval between each addition of liquid and centrifugation is 0.5-1 minute, the centrifugal force is 1500g, and the centrifugation time is 10-30 seconds, the same below; then use 50-80 μL 80 -85% acetonitrile aqueous solution was washed once, and the solution was removed by centrifugation. After the desalted N-glycan sample (DP7:BSA peptide = 1:1000) was lyophilized, it was redissolved with 80-160 μL of acetonitrile / trifluoroacetic acid / water solution, so that the volume fraction of acetonitrile in the solution after the reaction was 80-85 %, the volume fraction of TFA is 0-0.1%, the sample is loaded, and the solution is removed by centrifugation. Use 50-100 μL 75-85% acetonitrile / water solution to wash 4-6...

Embodiment 2

[0042] High-speed enzymatic digestion and solid-phase enrichment on cotton for N-glycoprotein-containing samples

[0043] Weigh 3-7 mg of absorbent cotton material and place it at the bottom of the centrifuge tube. After freeze-drying the N-glycoprotein sample to be processed (ASF:BSA enzymatic peptide = 1:50), redissolve it with 20-40 μL 25mM ammonium bicarbonate buffer to make the final concentration 10ng / μL-1000ng / μL , transferred to a centrifuge tube containing absorbent cotton material. Treat in a water bath at 100 degrees Celsius for 5 minutes, add 0.5 μL PNGase F enzyme after cooling, and enzymatically hydrolyze at 45-55 degrees Celsius for 0.5-1 hour. A certain volume of acetonitrile / trifluoroacetic acid / water solution is added to the solution after enzymolysis, so that the volume fraction of acetonitrile in the solution is 80-85%, the volume fraction of TFA is 0-0.1%, and the volume is 80-160 μL. Fill the pipette tip with absorbent cotton material and place it on th...

Embodiment 3

[0045] High-speed enzymatic digestion and solid-phase enrichment on cotton for complex samples of human serum

[0046] Weigh 3-7 mg of absorbent cotton material and place it at the bottom of the centrifuge tube. 1 μL of serum to be treated was diluted with 20-40 μL of 25 mM ammonium bicarbonate buffer, and then transferred to a centrifuge tube containing absorbent cotton material. Treat in a water bath at 100 degrees Celsius for 5 minutes, add 0.5 μL PNGase F enzyme after cooling, and enzymatically hydrolyze at 45-55 degrees Celsius for 0.5-1 hour. A certain volume of acetonitrile / trifluoroacetic acid / water solution is added to the solution after enzymolysis, so that the volume fraction of acetonitrile in the solution is 80-85%, the volume fraction of TFA is 0-0.1%, and the volume is 80-160 μL. Fill the pipette tip with absorbent cotton material and place it on the centrifuge tube. Load the remaining solution, centrifuge to remove the solution, the interval between each addi...

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Abstract

The invention belongs to the field of the glycoproteomics and glycomics, and relates to a method for rapid enzymolysis release, solid phase concentration and mass spectrometry of N-glycan. The methodcomprises the following steps of absorbing a solution containing a glycoprotein sample by using a degreasing cotton material, and performing high-temperature rapid PNGase F enzymolysis on the cotton to release N-glycan; adjusting solution environment after the enzymolysis is finished, and adsorbing free glycan on the degreasing cotton material by utilizing the high-density hydroxy and hydrogen bond of the glycan and like effects on the surface of the degreasing cotton material, and then cleaning and removing non-glycan molecules such as protein, polypeptide and like which are not bonded with the degreasing cotton material, and finally eluting the adsorbed glycan with the aqueous solution, and feeding into mass spectrometry. Through the method disclosed by the invention, the used material is cheap and easy to obtain, the preparation is easy, the operation is simple and convenient, the high-speed enzymolysis of the N-glycoprotein and the high-selective concentration and high-sensitive mass spectrometry of the N-glycan can be realized.

Description

technical field [0001] The invention belongs to the field of glycoproteomics and glycomics analysis, and relates to a method for rapid enzymatic release, solid-phase enrichment and mass spectrometry analysis of N-sugar chains in glycoproteins. The method of the present invention has higher selectivity and higher analytical sensitivity than the currently commonly used N-sugar chain enrichment and mass spectrometry methods, and can significantly shorten the time required to obtain sugar chains from serum and other samples, and has the advantages of simple steps , Easy and fast operation. Background technique [0002] The prior art discloses that protein N-glycosylation is one of the most common post-translational modifications in organisms. According to research reports, more than 50% of proteins in mammals can undergo N-glycosylation modification. Studies have shown that glycosylation has important biological significance. N-sugar chains connected to proteins play an importa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6851
Inventor 陆豪杰张莹彭叶
Owner FUDAN UNIV
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