Simultaneous quantification of N- and O-glycans using a solid-phase method

Nature Protocols
Shuang YangHui Zhang

Abstract

Glycosylation has a pivotal role in a diverse range of biological activities, modulating the structure and function of proteins. Glycogens coupled to the nitrogen atom (N-linked) of asparagine side chains or to the oxygen atom (O-linked) of serine and threonine side chains represent the two major protein glycosylation forms. N-glycans can be released by glycosidases, whereas O-glycans are often cleaved by chemical reaction. However, it is challenging to combine these enzymatic and chemical reactions in order to analyze both N- and O-glycans. We recently developed a glycoprotei n immobilization for glycan extraction (GIG) method that allows for the simultaneous analysis of N- and O-glycans on a solid support. GIG enables quantitative analysis of N-glycans and O-glycans from a single specimen and can be applied to a high-throughput automated platform. Here we provide a step-by-step GIG protocol that includes procedures for (i) protein immobilization on an aldehyde-active solid support by reductive amination; (ii) stabilization of fragile sialic acids by carbodiimide coupling; (iii) release of N-glycans by PNGase F digestion; (iv) release of O-glycans by β-elimination using ammonia in the presence of 1-phenyl-3-methyl-5-pyrazolone...Continue Reading

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Citations

Dec 12, 2017·Mass Spectrometry Reviews·Qiwei ZhangJianjun Li
Feb 10, 2018·Proteomics. Clinical Applications·Shuang YangJohn Cipollo
Nov 20, 2018·Expert Review of Proteomics·Jan TkacPeter Kasak
Sep 24, 2019·Biotechnology and Bioengineering·Cheng-Yu ChungMichael J Betenbaugh
Jul 19, 2020·Nature Protocols·Weiming YangHui Zhang
Aug 3, 2019·International Journal of Molecular Sciences·Nicolas SmargiassoCatherine Navarre
Jan 20, 2021·Cancer Treatment and Research Communications·Shuang YangShuwei Li
Jun 25, 2020·Molecular & Cellular Proteomics : MCP·Daniel G Delafield, Lingjun Li
Feb 5, 2021·Rapid Communications in Mass Spectrometry : RCM·Taylor BattellinoHélène Perreault
Apr 14, 2018·Journal of the American Society for Mass Spectrometry·Shuang YangJohn Cipollo
Sep 7, 2019·Journal of Agricultural and Food Chemistry·Yu LuZhongfu Wang
Jun 26, 2018·Analytical Chemistry·Shuang YangJohn Cipollo
Mar 20, 2018·Chemical Reviews·L Renee RuhaakCarlito B Lebrilla
Jun 13, 2021·Genomics, Proteomics & Bioinformatics·José Alexandre FerreiraLúcio Lara Santos

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