Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry
Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry
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DOI：
10.1021/acs.analchem.6b05129
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发表时间：
2017-05-02
影响因子：
7.4
通讯作者：
Heck, Albert J. R.
中科院分区：
文献类型：
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作者：
Wang, Guanbo;de Jong, Rob N.;Heck, Albert J. R.
糖基化蛋白质分子量的测定受到糖基化蛋白质的理化特性和异质性的严重影响。糖基化影响十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）和尺寸排阻色谱（SEC）分析过程中的蛋白质迁移。标准电喷雾电离（ESI）-质谱法不提供直接的解决方案，因为该方法受到广泛分布的糖型的紧密间隔的电荷状态引起的离子信号的广泛干扰的阻碍。在这里，我们介绍了一个本地串联MS为基础的方法，使电荷状态分辨率和电荷分配的蛋白质离子，包括那些逃避质量分析在标准的MS条件下。使用这种方法，我们确定了两个模型糖蛋白的分子量，高度和异质糖基化的蛋白质CD 38和表皮生长因子受体（EGFR）的细胞外结构域，以及这些蛋白质与特定抗体复合的总体分子量和结合化学计量。
The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.