糖基化的位点特异性表征需要完整的糖肽分析,最近的努力集中在如何使用串联质谱(MS/MS)最好地询问糖肽。梁式碰撞激活,即,高能碰撞解离(HCD),是一种有价值的方法,但是逐步碰撞能量HCD(sceHCD)和具有HCD补充激活(EThcD)的电子转移解离已成为潜在更合适的替代品。sceHCD和EThcD已成功用于大规模的糖蛋白质组实验,但是他们每个人都有某种程度的妥协。大多数进展发生在N-糖蛋白质组学领域。人们对将这一进展扩展到O-糖蛋白质组学越来越感兴趣,这需要比较两类糖肽的方法性能。这里,我们系统地探讨了传统HCD的优缺点,sceHCD,ETD,和EThcD用于完整的糖肽分析,并确定它们对N-和O-糖蛋白组应用的适用性。对于N-糖肽,HCD和sceHCD产生相似数量的标识,虽然sceHCD通常提供更高质量的光谱。两者在识别方面都显著优于EThcD方法,表明基于ETD的方法对于常规N-糖蛋白质组学是不需要的,即使它们可以产生更高质量的光谱。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,O-糖肽不能用足以用于N-糖肽的以HCD为中心的方法进行稳健表征,和旨在表征O-糖肽的糖蛋白质组学方法必须相应地构建。
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.