Optimal Dissociation Methods Differ for N- and O-Glycopeptides
Optimal Dissociation Methods Differ for N- and O-Glycopeptides
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DOI：
10.1021/acs.jproteome.0c00218
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发表时间：
2020-08-07
影响因子：
4.4
通讯作者：
Bertozzi, Carolyn R.
中科院分区：
文献类型：
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作者：
Riley, Nicholas M.;Malaker, Stacy A.;Bertozzi, Carolyn R.
糖基化的位点特异性表征需要完整的糖肽分析，最近的工作集中在如何使用串联质谱（MS/MS）最好地询问糖肽。束型碰撞激活，即高能碰撞离解（HCD），一直是一种有价值的方法，但阶梯式碰撞能量离解（sceHCD）和电子转移离解与HCD补充激活（EThcD）已经成为潜在更合适的替代方法。sceHCD和EThcD在大规模糖蛋白组学实验中都取得了成功，但它们都有一定程度的妥协。大多数进展发生在n -糖蛋白组学领域。有越来越多的兴趣将这一进展扩展到o糖蛋白组学，这需要比较两类糖肽的方法性能。在这里，我们系统地探讨了常规HCD、sceHCD、ETD和EThcD在完整糖肽分析中的优缺点，并确定了它们在N-和o -糖蛋白组学应用中的适用性。对于n -糖肽，HCD和sceHCD产生相似数量的鉴定，尽管sceHCD通常提供更高质量的光谱。两者在鉴定方面都明显优于EThcD方法，这表明基于etd的方法即使可以产生更高质量的光谱，也不需要常规的n -糖蛋白组学。相反，基于etd的方法，特别是EThcD，对于o -糖肽的位点特异性分析是必不可少的。我们的数据表明，用足以用于n -糖肽的以hcd为中心的方法无法对o -糖肽进行强有力的表征，因此必须相应地构建旨在表征o -糖肽的糖蛋白组学方法。
Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area of N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods in terms of identifications, indicating that ETD-based methods are not required for routine N-glycoproteomics even if they can generate higher quality spectra. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.