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Structural analysis of N- and O-glycans released from glycoproteins

Journal article
Authors P. H. Jensen
Niclas G. Karlsson
D. Kolarich
N. H. Packer
Published in Nature Protocols
Volume 7
Issue 7
Pages 1299-1310
ISSN 1754-2189
Publication year 2012
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 1299-1310
Language en
Links dx.doi.org/10.1038/nprot.2012.063
Keywords liquid chromatography/mass spectrometry, mass-spectrometry, linked, oligosaccharide, glycosylation, ms, glycomics, disease, cells, identification, profiles
Subject categories Medical Biotechnology

Abstract

This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive beta-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.

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