Mass spectrometry-based glycomics strategy for exploring N-linked glycosylation in eukaryotes and bacteria.
N-Glycosylation of proteins is recognized as one of the most common posttranslational modifications in eukaryotes. To date, most glycomics techniques are limited to examining eukaryotic pathways. Technologies capable of characterizing newly described N-linked glycosylation systems in bacteria from biologically relevant samples in an accurate, rapid, and cost-effective manner are needed. In this paper, a new glycomics strategy, based on the combination of nonspecific proteolytic digestion and permethylation, was devised that can be used for both eukaryotic and bacterial glycoproteins. Eukaryotic glycoproteins were digested with Pronase E with a higher ratio (enzyme/protein, 2:1-3:1) and a longer reaction time (48-72 h). The Asn-glycans were then purified using porous graphitic carbon cartridges followed by permethylation. The mass spectrometric data indicated that hydroxyl groups were methylated, while the amino group in asparagine underwent beta-elimination. Both modifications were evident from an increase of 111 Da in the molecular masses of permethylated Asn-glycans compared to the corresponding free oligosaccharides. NMR spectroscopy corroborated these results by showing that the mass difference resulted from beta-elimination of the free amino group in the asparagine residue. The method was validated with the characterization of the N-linked glycoproteins in total protein extracts from the bacterium Campylobacter jejuni 11168H. In addition to detecting the Asn-linked bacterial heptasaccharide, we also observed an unexpected free heptasaccharide intermediate that required a functional glycosylation pathway. These results demonstrate the usefulness of this method for screening N-linked glycans expressed by eukaryotes and bacteria and for detecting novel intermediates of N-linked glycosylation pathways.
Liu
Xin
X
NRC Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada K1A 0R6.
McNally
David J
DJ
Nothaft
Harald
H
Szymanski
Christine M
CM
Brisson
Jean-Robert
JR
Li
Jianjun
J
D000818:Animals; D016123:Campylobacter jejuni / Q000737:chemistry* / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000737:chemistry* / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods*; D053858:Metabolic Networks and Pathways; D015394:Molecular Structure; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry*; D011402:Pronase / Q000378:metabolism
D006020:Glycopeptides; D011134:Polysaccharides; D011402:Pronase