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

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...

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

Images