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 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, indicating that ETD-based methods are not required for routine N-glycoproteomics. 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.

糖基化的位点特异性表征需要完整的糖肽分析，并且最近的工作集中在如何使用串联质谱法（MS / MS）最佳地询问糖肽上。束型碰撞活化，即高能碰撞解离（HCD），已经成为一种有价值的方法，但是逐步碰撞能HCD（sceHCD）和电子转移离解与HCD辅助活化（EThcD）已成为潜在的更合适的替代方法。sceHCD和EThcD都已成功用于大规模糖蛋白组学实验，但是它们各自都会产生一定程度的折衷。大多数进展发生在N-糖代谢组学领域。越来越多的兴趣将这一进展扩展到O-糖蛋白组学领域，这需要对两种糖肽类的方法性能进行比较。这里，我们系统地探讨了完整的糖肽分析中常规HCD，sceHCD，ETD和EThcD的优缺点，并确定了它们对于N-和O-糖脂应用的适用性。对于N-糖肽，HCD和sceHCD产生的识别数相似，尽管sceHCD通常提供更高质量的光谱。两种方法均明显优于EThcD方法，这表明常规N-糖代谢组学不需要基于ETD的方法。相反，基于ETD的方法，尤其是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，不能以足以用于N-糖肽的HCD中心方法对O-糖肽进行稳健的表征，必须相应构建旨在表征O-糖肽的糖蛋白组学方法。