Kweon Hye Kyong, Håkansson Kristina
Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, USA.
J Proteome Res. 2008 Feb;7(2):749-55. doi: 10.1021/pr070386d. Epub 2008 Jan 3.
Gas-phase ion-electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), are advantageous for characterization of protein posttranslational modifications (PTMs), because labile modifications are not lost during the fragmentation process. However, at least two positive charges and relatively abundant precursor ions are required for ECD due to charge reduction and lower fragmentation efficiency compared to conventional gas-phase fragmentation techniques. Both these criteria are difficult to fulfill for phosphopeptides due to their acidic character. The negative ion mode operation of EDD is more compatible with phosphopeptide ionization, but EDD suffers from a fragmentation efficiency even lower than that of ECD. Recently, metal oxides such as ZrO 2 and TiO 2 have been shown to provide selective enrichment of phosphopeptides from proteolytic digests. Here, we utilize this enrichment strategy to improve ECD and EDD of phosphopeptides. This approach allowed determination of the locations of phosphorylation sites in highly acidic, multiply phosphorylated peptides from complex peptide mixtures by ECD. For singly phosphorylated peptides, EDD provided complementary sequence information compared to ECD.
气相离子 - 电子反应，包括电子捕获解离（ECD）和电子脱离解离（EDD），对于蛋白质翻译后修饰（PTM）的表征具有优势，因为在碎片化过程中不稳定的修饰不会丢失。然而，与传统的气相碎片化技术相比，由于电荷减少和碎片化效率较低，ECD至少需要两个正电荷和相对丰富的前体离子。由于磷酸肽的酸性特征，这两个标准对于磷酸肽来说都难以满足。EDD的负离子模式操作与磷酸肽电离更兼容，但EDD的碎片化效率甚至低于ECD。最近，已证明诸如ZrO₂和TiO₂等金属氧化物能够从蛋白水解消化物中选择性富集磷酸肽。在此，我们利用这种富集策略来改善磷酸肽的ECD和EDD。这种方法能够通过ECD确定复杂肽混合物中高度酸性、多磷酸化肽的磷酸化位点位置。对于单磷酸化肽，与ECD相比，EDD提供了互补的序列信息。
