Ma Cheng, Zhang Qi, Qu Jingyao, Zhao Xinyuan, Li Xu, Liu Yunpeng, Wang Peng George
Center for Diagnostics & Therapeutics, Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States.
Department of Parasitology, Faculty of Biology, Philipps University Marburg, Marburg, Hessen 35043, Germany.
J Proteomics. 2015 Jan 30;114:61-70. doi: 10.1016/j.jprot.2014.09.001. Epub 2014 Sep 8.
The core fucosylation (CF) of N-glycoproteins plays important roles in regulating protein functions during biological development, and it has also been shown to be up-regulated in several high metastasis cancer cell lines. Therefore, global profiling and quantitative characterization of CF-glycoproteins may reveal potent biomarkers for clinical applications. However, due to the complex fragmentation pattern of CF-glycopeptides, accurately identifying CF-glycosylation sites via mass spectrometry with high throughput remains a formidable challenge. In this study, we established a precise CF-glycosylation site identification strategy with UHPLC LTQ-Orbitrap Elite under low- and high-normalized collision energy (LHNCE) conditions. To demonstrate the feasibility of LHNCE, the CF-glycopeptides of target proteins in clinical plasma samples were applied and compared as a preliminary demonstration and resulted in the assignment of 357 unique CF-glycosylation sites from 209 CF-glycoproteins. In this study, the largest human plasma CF-glycosylation site database was constructed, and at least three-fold more CF-sites were identified compared to previously published studies. The results further demonstrated that LHNCE provides an important approach for CF-glycoprotein function studies and biomarker screening in cancer research.
Core-fucosylation (CF) is a kind of N-linked glycosylation in which an α1,6-linked fucose is added to the innermost N-acetylglucosamine (GlcNAc) residue. It has been proved that core-fucosylation is involved in regulating biological processes in mammals. Abnormal core-fucosylation has been demonstrated in human pathological processes, such as metastasis. For example, the CF-glycosylation of an α-fetoprotein isoform (AFP-L3) was approved as a biomarker of hepatocellular carcinoma (HCC). In addition, GP73 is also a well-known biomarker and its CF-glycosylation level will increase in liver cancer patients. Therefore, it is crucial to develop a strategy for mapping human CF-glycosylation.
N-糖蛋白的核心岩藻糖基化（CF）在生物发育过程中调节蛋白质功能方面发挥着重要作用，并且在几种高转移癌细胞系中也被证明上调。因此，CF-糖蛋白的全局分析和定量表征可能揭示临床应用中有潜力的生物标志物。然而，由于CF-糖肽复杂的碎裂模式，通过质谱高通量准确鉴定CF-糖基化位点仍然是一项艰巨的挑战。在本研究中，我们在低和高归一化碰撞能量（LHNCE）条件下，利用超高效液相色谱-线性离子阱-轨道阱高分辨质谱仪（UHPLC LTQ-Orbitrap Elite）建立了一种精确的CF-糖基化位点鉴定策略。为证明LHNCE的可行性，应用并比较了临床血浆样本中靶蛋白的CF-糖肽作为初步验证，结果从209种CF-糖蛋白中鉴定出357个独特的CF-糖基化位点。在本研究中，构建了最大的人血浆CF-糖基化位点数据库，与先前发表的研究相比鉴定出的CF-位点至少多三倍。结果进一步证明LHNCE为癌症研究中CF-糖蛋白功能研究和生物标志物筛选提供了一种重要方法。
核心岩藻糖基化（CF）是一种N-连接糖基化，其中一个α1,6-连接的岩藻糖被添加到最内层的N-乙酰葡糖胺（GlcNAc）残基上。已证明核心岩藻糖基化参与调节哺乳动物的生物过程。在人类病理过程如转移中已证实存在异常的核心岩藻糖基化。例如，甲胎蛋白异构体（AFP-L3）的CF-糖基化被批准作为肝细胞癌（HCC）的生物标志物。此外，GP73也是一种著名的生物标志物，其CF-糖基化水平在肝癌患者中会升高。因此，开发一种绘制人类CF-糖基化图谱的策略至关重要。
