8-plex LC-MS/MS Analysis of Permethylated N-Glycans Achieved by Using Stable Isotopic lodomethane
8-plex LC-MS/MS Analysis of Permethylated N-Glycans Achieved by Using Stable Isotopic lodomethane
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DOI：
10.1021/acs.analchem.9b02411
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发表时间：
2019-09-17
影响因子：
7.4
通讯作者：
Mechref, Yehia
中科院分区：
文献类型：
--
作者：
Dong, Xue;Peng, Wenjing;Mechref, Yehia
糖基化是蛋白质翻译后的重要修饰。许多疾病，如癌症，已被证明与异常糖基化有关。高通量定量方法是近年来糖组学研究的热点。随着高分辨率质谱技术的发展，糖组学检测的灵敏度大大提高；然而，目前常用的MS-based技术大多集中在相对定量分析上，难以提供直接的比较糖糖定量结果。在本研究中，我们在LC-ESI-MS平台上建立了一种新的多元糖糖分析方法。在过甲基化过程中，使用碘甲烷试剂CH2DI、CHD2I、CD3I、(CH3I)-C-13、(CH2DI)-C-13、(CHD2I)-C-13、(CD3I)-C-13和CH3I对还原聚糖进行稳定同位素标记。在LC-MS的单次分析中，最多可以进行8-plex糖糖谱分析，并且100 k的质量分辨率足以允许8-plex标记聚糖之间的质量差异的基线分辨率。该方法的主要优点是克服了纳米esi引起的定量波动，便于准确反映样品中定量信息的比较定量分析水平，大大缩短了分析时间。定量验证了牛胎儿蛋白和模型糖蛋白混合物（RNase B、牛胎儿蛋白和IgG）所释放的聚糖具有良好的线性关系（R-2 = 0.9884），动态范围为0.1 ~ 10。8-plex策略成功地应用于癌细胞系的比较糖糖研究。结果表明，唾液化聚糖的不同分布与细胞系的转移特性有关，为更好地了解乳腺癌脑转移提供了重要线索。
Glycosylation is an important post-translational modification of proteins. Many diseases, such as cancer, have proved to be related to aberrant glycosylation. High throughput quantitative methods have gained attention recently in the study of glycomics. With the development of high-resolution mass spectrometry, the sensitivity of detection in glycomics has largely improved; however, most of the commonly used MS-based techniques are focused on relative quantitative analysis, which can hardly provide direct comparative glycomic quantitation results. In this study, we developed a novel multiplex glycomic analysis method on an LC-ESI-MS platform. Reduced glycans were stable isotopic labeled during the permethylation procedure, with the use of iodomethane reagents CH2DI, CHD2I, CD3I, (CH3I)-C-13, (CH2DI)-C-13, (CHD2I)-C-13, (CD3I)-C-13, and CH3I. Up to 8-plex glycomic profiling was possible in a single analysis by LC-MS, and a 100 k mass resolution was sufficient to allow a baseline resolution of the mass differences among the 8-plex labeled glycans. The major advantages of this method are that it overcomes quantitative fluctuations caused by nanoESI, it facilitates a level of comparative quantitative glycomic analysis that accurately reflects the quantitative information in samples, and it dramatically shortens analysis time. Quantitation validation was tested on glycans released from bovine fetuin and model glycoprotein mixtures (RNase B, bovine fetuin, and IgG) with good linearity (R-2 = 0.9884) and a dynamic range from 0.1 to 10. The 8-plex strategy was successfully applied to a comparative glycomic study of cancer cell lines. The results demonstrate that different distributions of sialylated glycans are related to the metastatic properties of cell lines and provide important clues for a better understanding of breast cancer brain metastasis.