Li Bensheng, An Hyun Joo, Kirmiz Crystal, Lebrilla Carlito B, Lam Kit S, Miyamoto Suzanne
Department of Chemistry, Biochemistry and Molecular Medicine, University of California, Davis, California 95616, USA.
J Proteome Res. 2008 Sep;7(9):3776-88. doi: 10.1021/pr800297u. Epub 2008 Jul 22.
Ovarian cancer is difficult to diagnose in women because symptoms of the disease are often not noticed until the disease has progressed to an advanced untreatable stage. Although a serum test, CA125, is currently available to assist with monitoring treatment of ovarian cancer, this test lacks the necessary specificity and sensitivity for early detection. Therefore, better biomarkers of ovarian cancer are needed. A glycoprotein analysis approach was undertaken using high resolution Fourier transform ion cyclotron resonance mass spectrometry to analyze glycosylated proteins present in the conditioned media of ovarian cancer cell lines and in sera obtained from ovarian cancer patients and normal controls. In this study, glycosylated proteins were separated by gel electrophoresis, and individual glycoproteins were selected for glycosylation analysis and protein identification. The attached glycans from each protein were released and profiled by mass spectrometry. Glycosylation of a mucin protein and a large glycosylated protein isolated from the ES2 ovarian cancer cell line was determined to consist of mostly O-linked glycans. Four prominent glycoproteins of approximate 517, 370, 250, 163 kDa from serum samples were identified as two forms of apolipoprotein B-100, fibronectin, and immunoglobulin A1, respectively. Mass spectrometric analysis of glycans isolated from apolipoprotein B-100 (517 kD) showed the presence of small, specific O-linked oligosaccharides. In contrast, analysis of fibronectin (250 kD) and immunoglobulin A1 (163 kD) produced N-linked glycan fragments in forms that were sufficiently different from the glycans obtained from the corresponding protein band present in the normal serum samples. This study shows that not only a single protein but several are aberrantly glycosylated, and those abnormal glycosylation changes can be detected and may ultimately serve as glycan biomarkers for ovarian cancer.
卵巢癌在女性中难以诊断，因为该疾病的症状往往直到病情发展到晚期无法治疗阶段才会被注意到。尽管目前有一种血清检测方法CA125可用于辅助监测卵巢癌的治疗，但该检测缺乏早期检测所需的特异性和敏感性。因此，需要更好的卵巢癌生物标志物。采用高分辨率傅里叶变换离子回旋共振质谱法进行糖蛋白分析，以分析卵巢癌细胞系条件培养基以及卵巢癌患者和正常对照血清中存在的糖基化蛋白。在本研究中，通过凝胶电泳分离糖基化蛋白，并选择单个糖蛋白进行糖基化分析和蛋白质鉴定。从每种蛋白质上释放附着的聚糖并通过质谱进行分析。从ES2卵巢癌细胞系分离出的一种粘蛋白和一种大的糖基化蛋白的糖基化被确定主要由O-连接聚糖组成。从血清样本中鉴定出四种分子量约为517、370、250、163 kDa的突出糖蛋白，分别为两种形式的载脂蛋白B-100、纤连蛋白和免疫球蛋白A1。对从载脂蛋白B-100（517 kD）分离出的聚糖进行质谱分析，显示存在小的特异性O-连接寡糖。相比之下，对纤连蛋白（250 kD）和免疫球蛋白A1（163 kD）的分析产生的N-连接聚糖片段形式与正常血清样本中相应蛋白条带获得的聚糖有足够差异。这项研究表明，不仅单一蛋白质而且多种蛋白质都存在异常糖基化，并且这些异常糖基化变化可以被检测到，最终可能作为卵巢癌的聚糖生物标志物。
