Glycomic/glycoproteomic analysis by liquid chromatography/mass spectrometry: Analysis of glycan structure alternation in cells
Glycomic/glycoproteomic analysis by liquid chromatography/mass spectrometry: Analysis of glycan structure alternation in cells
复制标题
DOI：
10.1002/pmic.200401330
复制
发表时间：
2005-12-01
期刊：
影响因子：
3.4
通讯作者：
Hayakawa, T
中科院分区：
文献类型：
--
作者：
Hashii, N;Kawasaki, N;Hayakawa, T
糖基转移酶表达的改变和随后寡糖结构的变化在几种疾病中均有报道。糖蛋白中聚糖结构改变的分析在发现治疗和诊断标志物中变得越来越重要。在这项研究中，我们提出了一种策略，糖/糖蛋白质组学分析的基础上，寡糖分析LC/MS其次是蛋白质组学方法，包括2-DE和2-D凝集素印迹。作为异常细胞的模型，我们使用了用N-乙酰葡糖胺转移酶III（GnT-III）转染的中国仓鼠卵巢细胞，GnT-III催化N-连接寡糖的甘露糖基核心的P-甘露糖中加入二等分N-乙酰葡糖胺（GlcNAc）。配备石墨化碳柱（GCC）的LC/ MS使我们能够阐明GnT-III表达诱导的结构改变。使用2-D凝集素印迹，然后使用LC/MS/MS，成功将GnT-III转染的细胞中携带额外N-乙酰己糖胺的蛋白质鉴定为整合素α 3。因此，通过GCC-LC/MS进行寡糖谱分析，然后采用蛋白质组学方法，可以成为糖组学/糖蛋白质组学分析的有力工具。
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 P-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 alpha 3. Thus, oligosaccharide profiling by GCC-LC/MS followed by proteomic methods can be a powerful tool for glycomic/glycoproteomic analysis.