Porras-Yakushi Tanya R, Sweredoski Michael J, Hess Sonja
California Institute of Technology, Beckman Institute, Pasadena, CA, 91125, USA.
J Am Soc Mass Spectrom. 2015 Sep;26(9):1580-7. doi: 10.1007/s13361-015-1168-0. Epub 2015 May 21.
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.
对泛素化修饰组进行全面分析是充分理解泛素化调控作用的前提条件。然而，关键质谱参数对泛素化修饰组分析的影响尚未得到充分探究。在本研究中，我们发现，单独使用电子转移解离（ETD）碎裂法或作为决策树方法的一部分，相比于传统的碰撞诱导解离（CID）或高能碰撞解离（HCD）方法，可使富含K-ε-GG肽段的样品中泛素化位点鉴定数量增加约2倍。主要观察到的前体离子为3 + 电荷或更高电荷的物种，质量范围在300 - 1200 m/z。使用N-乙基马来酰亚胺作为烷基化剂，以减少因卤代乙酰胺过度烷基化导致的假阳性鉴定。这些结果表明，除了缩小质量范围和使用N-乙基马来酰亚胺外，ETD碎裂法的应用比传统的CID和HCD分析能够产生更多高可信度的泛素化位点鉴定结果。
