Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.
We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.
Kaji
Hiroyuki
H
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan.
Saito
Haruna
H
Yamauchi
Yoshio
Y
Shinkawa
Takashi
T
Taoka
Masato
M
Hirabayashi
Jun
J
Kasai
Ken-ichi
K
Takahashi
Nobuhiro
N
Isobe
Toshiaki
T
D029742:Caenorhabditis elegans Proteins / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017124:Conserved Sequence; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D037102:Lectins / Q000737:chemistry; D046911:Macromolecular Substances; D008358:Mannose / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D029742:Caenorhabditis elegans Proteins; D006023:Glycoproteins; D037102:Lectins; D046911:Macromolecular Substances; D010455:Peptides; D011134:Polysaccharides; D008358:Mannose