School of BioSciences - Research Publications
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ItemCharacterization of protein N-glycosylation by tandem mass spectrometry using complementary fragmentation techniques(FRONTIERS MEDIA SA, 2015-08-28)The analysis of post-translational modifications (PTMs) by proteomics is regarded as a technically challenging undertaking. While in recent years approaches to examine and quantify protein phosphorylation have greatly improved, the analysis of many protein modifications, such as glycosylation, are still regarded as problematic. Limitations in the standard proteomics workflow, such as use of suboptimal peptide fragmentation methods, can significantly prevent the identification of glycopeptides. The current generation of tandem mass spectrometers has made available a variety of fragmentation options, many of which are becoming standard features on these instruments. We have used three common fragmentation techniques, namely CID, HCD, and ETD, to analyze a glycopeptide and highlight how an integrated fragmentation approach can be used to identify the modified residue and characterize the N-glycan on a peptide.
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ItemN-linked Glycan Micro-heterogeneity in Glycoproteins of Arabidopsis(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2018-03)N-glycosylation is one of the most common protein post-translational modifications in eukaryotes and has a relatively conserved core structure between fungi, animals and plants. In plants, the biosynthesis of N-glycans has been extensively studied with all the major biosynthetic enzymes characterized. However, few studies have applied advanced mass spectrometry to profile intact plant N-glycopeptides. In this study, we use hydrophilic enrichment, high-resolution tandem mass spectrometry with complementary and triggered fragmentation to profile Arabidopsis N-glycopeptides from microsomal membranes of aerial tissues. A total of 492 N-glycosites were identified from 324 Arabidopsis proteins with extensive N-glycan structural heterogeneity revealed through 1110 N-glycopeptides. To demonstrate the precision of the approach, we also profiled N-glycopeptides from the mutant (xylt) of β-1,2-xylosyltransferase, an enzyme in the N-glycan biosynthetic pathway. This analysis represents the most comprehensive and unbiased collection of Arabidopsis N-glycopeptides revealing an unsurpassed level of detail on the micro-heterogeneity present in N-glycoproteins of Arabidopsis. Data are available via ProteomeXchange with identifier PXD006270.