ETD Outperforms CID and HCD in the Analysis of the Ubiquitylated Proteome.
ETD Outperforms CID and HCD in the Analysis of the Ubiquitylated Proteome.
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DOI：
10.1007/s13361-015-1168-0
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发表时间：
2015-09
影响因子：
3.2
通讯作者：
Hess S
中科院分区：
文献类型：
--
作者：
Porras-Yakushi TR;Sweredoski MJ;Hess S
对泛素化组的全面分析是充分理解泛素化调控作用的前提。然而，关键的质谱参数对泛素分析的影响还没有得到充分的探讨。在这项研究中，我们表明，使用电子转移解离（ETD）断裂，无论是专门或作为决策树方法的一部分，导致约。2-与传统碰撞诱导解离（CID）或高能碰撞解离（HCD）方法相比，K-ε-GG肽富集样品中泛素化位点鉴定增加了一倍。前体离子主要被观察为3+带电物质或更高，质量范围为300-1200 m/z。N-乙基马来酰亚胺用作烷基化剂以减少由卤代乙酰胺过度烷基化引起的假阳性鉴定。这些结果表明，除了缩小质量范围和使用N-乙基马来酰亚胺之外，ETD片段化的应用产生比常规CID和HCD分析更高置信度的泛素化位点鉴定。
Comprehensive analysis of the ubiquitylome is a prerequisite to fully understand the regulatory role of ubiquitylation. However, the impact of key mass spectrometry parameters on ubiquitylome analyses has not been fully explored. In this study, we show that using electron transfer dissociation (ETD) fragmentation, either exclusively or as part of a decision tree method, leads to ca. 2-fold increase in ubiquitylation site identifications in K-ε-GG peptide-enriched samples over traditional collisional-induced dissociation (CID) or higher-energy collision dissociation (HCD) methods. Precursor ions were predominantly observed as 3+ charged species or higher and in a mass range 300–1200 m/z. N-ethylmaleimide was used as an alkylating agent to reduce false positive identifications resulting from overalkylation with halo-acetamides. These results demonstrate that the application of ETD fragmentation, in addition to narrowing the mass range and using N-ethylmaleimide yields more high-confidence ubiquitylation site identification than conventional CID and HCD analysis.