Nature Biotechnology
21, 667 - 672 (2003)
Published online: 18 May 2003; | doi:10.1038/nbt829
Lectin affinity capture, isotope-coded tagging and mass spectrometry
to identify N-linked glycoproteinsHiroyuki Kaji1, Haruna Saito1, Yoshio Yamauchi2, Takashi Shinkawa1, Masato Taoka1, Jun Hirabayashi3, Ken-ichi Kasai3, Nobuhiro Takahashi2, 4
& Toshiaki Isobe1, 2, 51
Department of Chemistry, Graduate School of
Science, Tokyo Metropolitan University, Hachioji,
Tokyo 192-0397, Japan. 2
The Integrated Proteomics System Project, Pioneer
Research on Genome the Frontier, MEXT, c/o Tokyo Metropolitan University,
Hachioji, Tokyo 192-0397,
Japan. 3
Department of Biological Chemistry, Faculty of
Pharmaceutical Science, Teikyo University, Sagamiko,
Kanagawa 199-0195, Japan. 4
Department of Applied Biological Science, and
Department of Biotechnology, United Graduate School of Agriculture, Tokyo
University of Agriculture & Technology, Fuchu,
Tokyo 183-8509, Japan. 5
Proteomics Division, Medical Research Institute,
The University of Tokyo, Minato-ku, Tokyo
108-8639, Japan.
Correspondence should be addressed to Toshiaki Isobe isobe-toshiaki@c.metro-u.ac.jpWe 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.
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