Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging.
Protein post-translational modifications (PTMs), such as glycosylation and phosphorylation, are crucial for various signaling and regulatory events, and are therefore an important objective of proteomics research. We describe here a protocol for isotope-coded glycosylation site-specific tagging (IGOT), a method for the large-scale identification of N-linked glycoproteins from complex biological samples. The steps of this approach are: (1) lectin column-mediated affinity capture of glycopeptides generated by protease digestion of protein mixtures; (2) purification of the enriched glycopeptides by hydrophilic interaction chromatography (HIC); (3) peptide-N-glycanase-mediated incorporation of a stable isotope tag, 18O18O, specifically at the N-glycosylation site; and (4) identification of 18O-tagged peptides by liquid chromatography-coupled mass spectrometry (LC/MS)-based proteomics technology. The application of this protocol to the characterization of N-linked glycoproteins from crude extracts of the nematode Caenorhabditis elegans or mouse liver provides a list of hundreds to a thousand glycoproteins and their sites of glycosylation within a week.
Kaji
Hiroyuki
H
Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji, Tokyo 192-0397, Japan.
Yamauchi
Yoshio
Y
Takahashi
Nobuhiro
N
Isobe
Toshiaki
T
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D007553:Isotope Labeling / Q000379:methods*; D037102:Lectins / Q000737:chemistry; D012107:Research Design; D053719:Tandem Mass Spectrometry
D006020:Glycopeptides; D037102:Lectins