Glycomic/glycoproteomic analysis by liquid chromatography/mass spectrometry: analysis of glycan structural alteration in cells.
The alteration of glycosyltransferase expression and the subsequent changes in oligosaccharide structures are reported in several diseases. The analysis of glycan structural alteration in glycoproteins is becoming increasingly important in the discovery of therapies and diagnostic markers. In this study, we propose a strategy for glycomic/glycoproteomic analysis based on oligosaccharide profiling by LC/MS followed by proteomic approaches, including 2-DE and 2-D lectin blot. As a model of aberrant cells, we used Chinese hamster ovary cells transfected with N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) to beta-mannose of the mannosyl core of N-linked oligosaccharides. LC/MS equipped with a graphitized carbon column (GCC) enabled us to elucidate the structural alteration induced by the GnT-III expression. Using 2-D lectin blot followed by LC/MS/MS, the protein carrying an extra N-acetylhexosamine in cells transfected with GnT-III was successfully identified as integrin alpha3. Thus, oligosaccharide profiling by GCC-LC/MS followed by proteomic methods can be a powerful tool for glycomic/glycoproteomic analysis.
Hashii
Noritaka
N
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Kawasaki
Nana
N
Itoh
Satsuki
S
Hyuga
Mashashi
M
Kawanishi
Toru
T
Hayakawa
Takao
T
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002460:Cell Line; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006031:Glycosylation; D006108:Graphite; D006801:Humans; D037102:Lectins / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012995:Solubility
D037102:Lectins; D011134:Polysaccharides; D006108:Graphite