Site-specific protein glycosylation analysis with glycan isomer differentiation |
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Authors: | Serenus Hua Charles C Nwosu John S Strum Richard R Seipert Hyun Joo An Angela M Zivkovic J Bruce German Carlito B Lebrilla |
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Institution: | (1) Department of Chemistry, University of California, Davis, CA 95616, USA;(2) Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764, South Korea;(3) Department of Food Science and Technology, University of California, Davis, CA 95616, USA;(4) Foods for Health Institute, University of California, Davis, CA 95616, USA;(5) Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA; |
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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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