Hashii Noritaka, Kawasaki Nana, Itoh Satsuki, Hyuga Mashashi, Kawanishi Toru, Hayakawa Takao
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Proteomics. 2005 Dec;5(18):4665-72. doi: 10.1002/pmic.200401330.
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.
在多种疾病中均报道了糖基转移酶表达的改变以及随后寡糖结构的变化。糖蛋白中聚糖结构改变的分析在治疗方法的发现和诊断标志物的研究中变得越来越重要。在本研究中，我们提出了一种基于液相色谱/质谱联用（LC/MS）进行寡糖谱分析，随后采用蛋白质组学方法（包括二维电泳（2-DE）和二维凝集素印迹）的糖组学/糖蛋白质组学分析策略。作为异常细胞的模型，我们使用了转染了N-乙酰葡糖胺基转移酶III（GnT-III）的中国仓鼠卵巢细胞，该酶催化将一个平分型N-乙酰葡糖胺（GlcNAc）添加到N-连接寡糖甘露糖核心的β-甘露糖上。配备石墨化碳柱（GCC）的LC/MS使我们能够阐明由GnT-III表达诱导的结构改变。通过二维凝集素印迹，随后进行LC/MS/MS，成功鉴定出在转染了GnT-III的细胞中携带额外N-乙酰己糖胺的蛋白质为整合素α3。因此，通过GCC-LC/MS进行寡糖谱分析，随后采用蛋白质组学方法，可成为糖组学/糖蛋白质组学分析的有力工具。
