Institute of Molecular Biology, National Chung Hsing University, No. 250, Kuo-Kuang Road, Taichung 402, Taiwan.
J Am Soc Mass Spectrom. 2011 Jan;22(1):57-66. doi: 10.1007/s13361-010-0020-9. Epub 2011 Jan 27.
Electron-transfer dissociation (ETD) is a useful peptide fragmentation technique that can be applied to investigate post-translational modifications (PTMs), the sequencing of highly hydrophilic peptides, and the identification of large peptides and even intact proteins. In contrast to traditional fragmentation methods, such as collision-induced dissociation (CID), ETD produces c- and z(·)-type product ions by randomly cleaving the N-Cα bonds. The disappointing fragmentation efficiency of ETD for doubly charged peptides and phosphopeptide ions has been improved by ETcaD (supplemental activation). However, the ETD data derived from most database search algorithms yield low confidence scores due to the presence of unreacted precursors and charge-reduced ions within MS/MS spectra. In this work, we demonstrate that eight out of ten standard doubly charged peptides and phosphopeptides can be effortlessly identified by electron-transfer coupled with collision-induced dissociation (ET/CID) using the SEQUEST algorithm without further spectral processing. ET/CID was performed with the further dissociation of the charge-reduced ions isolated from ETD ion/ion reactions. ET/CID had high fragmentation efficiency, which elevated the confidence scores of doubly charged peptide and phosphospeptide sequencing. ET/CID was found to be an effective fragmentation strategy in "bottom-up" proteomic analysis.
电子转移解离（ETD）是一种有用的肽片段化技术，可用于研究翻译后修饰（PTMs）、高度亲水肽的测序以及大肽甚至完整蛋白质的鉴定。与传统的片段化方法（如碰撞诱导解离（CID））相比，ETD 通过随机裂解 N-Cα 键产生 c- 和 z(·)-型产物离子。通过补充激活（ETcaD），提高了 ETD 对双电荷肽和磷酸肽离子的碎片化效率。然而，由于 MS/MS 谱中存在未反应的前体和电荷降低的离子，大多数数据库搜索算法生成的 ETD 数据的置信分数较低。在这项工作中，我们证明了 SEQUEST 算法在无需进一步光谱处理的情况下，通过电子转移与碰撞诱导解离（ET/CID）的结合，能够轻松识别十种标准双电荷肽和磷酸肽中的八种。对从 ETD 离子/离子反应中分离出的电荷降低的离子进行进一步的解离，以进行 ET/CID。ET/CID 具有很高的片段化效率，提高了双电荷肽和磷酸肽测序的置信分数。ET/CID 被发现是“自上而下”蛋白质组分析中的一种有效的片段化策略。
