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Mass Spectrometric Analysis of Glycosphingolipid Antigens
Published on: April 16, 2013
Glycomic/glycoproteomic analysis by liquid chromatography/mass spectrometry: analysis of glycan structural alteration
Noritaka Hashii1, Nana Kawasaki, Satsuki Itoh
1Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Abstract:
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.
Insights
Glycosyltransferase alterations impact disease. This study introduces a glycomic/glycoproteomic strategy using LC/MS and proteomics to identify altered glycoproteins like integrin alpha3 in transfected cells.
Area of Science:
- Glycomics and Glycoproteomics
- Biochemistry and Molecular Biology
Background:
- Altered glycosyltransferase expression and resulting changes in oligosaccharide structures are linked to various diseases.
- Analyzing glycan structural alterations in glycoproteins is crucial for developing new therapies and diagnostic markers.
Purpose of the Study:
- To develop and validate a strategy for glycomic/glycoproteomic analysis.
- To investigate structural alterations in N-linked oligosaccharides induced by N-acetylglucosaminyltransferase III (GnT-III) expression.
Main Methods:
- Oligosaccharide profiling using liquid chromatography/mass spectrometry (LC/MS) with a graphitized carbon column (GCC).
- Proteomic analysis including 2-DE and 2-D lectin blot.
- LC/MS/MS for structural elucidation and protein identification.
Main Results:
- GCC-LC/MS successfully elucidated structural alterations in oligosaccharides due to GnT-III expression.
- 2-D lectin blot combined with LC/MS/MS identified integrin alpha3 as a protein with altered glycosylation (addition of bisecting N-acetylglucosamine).
Conclusions:
- The proposed strategy combining oligosaccharide profiling and proteomic methods is effective for glycomic/glycoproteomic analysis.
- This approach can identify specific glycoproteins with aberrant glycosylation patterns, aiding in disease marker discovery.
