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Citation
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HERO ID
752189
Reference Type
Journal Article
Title
Site-specific protein glycosylation analysis with glycan isomer differentiation
Author(s)
Hua, S; Nwosu, CC; Strum, JS; Seipert, RR; An, HJ; Zivkovic, AM; German, JB; Lebrilla, CB
Year
2012
Is Peer Reviewed?
Yes
Journal
Analytical and Bioanalytical Chemistry
ISSN:
1618-2642
EISSN:
1618-2650
Volume
403
Issue
5
Page Numbers
1291-1302
Language
English
PMID
21647803
DOI
10.1007/s00216-011-5109-x
Abstract
Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.
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Nanoscale Carbon
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