Analysis of singly glycosylated peptides has evolved to a point where large-scale LC-MS analyses can be performed at almost the same scale as proteomics experiments. While collisionally activated dissociation (CAD) remains the mainstay of bottom-up analyses, it performs poorly for the middle-down analysis of multiply glycosylated peptides. With improvements in instrumentation, electron-activated dissociation (ExD) modes are becoming increasingly prevalent for proteomics experiments and for the analysis of fragile modifications such as glycosylation. While these methods have been applied for glycopeptide analysis in isolated studies, an organized effort to compare their efficiencies, particularly for analysis of multiply glycosylated peptides (termed here middle-down glycoproteomics), has not been made. We therefore compared the performance of different ExD modes for middle-down glycopeptide analyses. We identified key features among the different dissociation modes and show that increased electron energy and supplemental activation provide the most useful data for middle-down glycopeptide analysis. Graphical Abstract.

中文翻译：

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多层糖基化肽的中下分析的碰撞和电子解离模式的比较。

单糖基化肽段的分析已经发展到可以进行与蛋白质组学实验几乎相同规模的大规模LC-MS分析的程度。尽管碰撞激活解离（CAD）仍然是自下而上分析的主要手段，但对于糖基化多肽的中下分析却表现不佳。随着仪器的改进，电子活化解离（ExD）模式在蛋白质组学实验和易碎修饰（如糖基化）分析中正变得越来越普遍。尽管这些方法已用于分离研究中的糖肽分析，但尚未做出有组织的努力来比较它们的效率，特别是用于分析多个糖基化肽（此处称为中-下糖蛋白组学）的努力。因此，我们比较了中下糖肽分析的不同ExD模式的性能。我们确定了不同的解离模式之间的关键特征，并表明增加的电子能量和补充激活为中下糖肽分析提供了最有用的数据。图形概要。