期刊
NATURE PROTOCOLS
卷 12, 期 6, 页码 1229-1244出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.034
关键词
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资金
- National Institutes of Health, National Cancer Institute
- Early Detection Research Network (EDRN) [U01CA152813]
- Clinical Proteomic Tumor Analysis Consortium (CPTAC) [U24CA160036]
- National Institutes of Health, National Heart Lung and Blood Institute Programs of Excellence in Glycosciences [P01HL107153]
- Johns Hopkins Proteomics Center [N01-HV-00240]
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 II-elimination using ammonia in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP) to prevent alditol peeling from O-glycans; (v) mass spectrometry (MS) analysis; and (vi) data analysis for identification of glycans using in-house developed software (GIG Tool; free to download via http://www.biomarkercenter.org/gigtool). The GIG tool extracts precursor masses, oxonium ions and glycan fragments from tandem (liquid chromatography (LC)-MS/MS) mass spectra for glycan identification, and reporter ions from quatemary amine containing isobaric tag for glycan (QUANTITY) isobaric tags are used for quantification of the relative abundance of N-glycans. The GIG protocol takes similar to 3 d.
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