Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation
Development of a combined chemical and enzymatic approach for the mass spectrometric identification and quantification of aberrant N-glycosylation
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DOI：
10.1016/j.jprot.2011.12.015
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发表时间：
2012-02-16
影响因子：
3.3
通讯作者：
Zou, Hanfa
中科院分区：
文献类型：
--
作者：
Chen, Rui;Wang, Fangjun;Zou, Hanfa
异常蛋白质糖基化的直接质谱分析是对现有分析技术的挑战。除了凝集素亲和层析，没有其他糖基化富集技术可用于分析异常糖基化。在这项研究中，我们开发了一种结合化学和酶的策略作为一种替代的质谱分析异常糖基化。用反向糖印迹富集唾液酸化糖肽，通过内切糖苷酶F3切割，并通过质谱法分析，其中中性丢失触发MS 3在碰撞诱导解离（CID）和电子转移解离（ETD）中。有趣的是，所得到的糖肽的很大一部分被发现与N-乙酰葡糖胺（N-GlcNAc）连接的岩藻糖，这表明鉴定了携带末端唾液酸化和核心岩藻糖基化的异常糖基化。在肝细胞癌（HCC）患者血清样本中共鉴定出69个异常N-糖基化位点。鉴定后，还使用稳定同位素二甲基标记对这种异常糖基化的水平进行定量，并比较来自肝硬化和HCC的合并血清样品。6个糖基化位点表现出较高的异常水平，这表明我们开发的策略在异常糖基化的定性和定量研究中是有效的。(C)2011爱思唯尔有限公司版权所有。
Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS3 in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylaton that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular carcinoma (HCC) patients. Following the identification, quantification of the level of this aberrant glycosylation was also carried out using stable isotope dimethyl labeling and pooled sera sample from liver cirrhosis and HCC was compared. Six glycosylation sites demonstrated elevated level of aberrancy, which demonstrated that our developed strategy was effective in both qualitative and quantitative studies of aberrant glycosylation. (C) 2011 Elsevier B.V. All rights reserved.