Department of Chemistry, University of California, Riverside, California 92521, USA.
Anal Chem. 2011 Sep 1;83(17):6818-26. doi: 10.1021/ac201647w. Epub 2011 Aug 8.
Identification of phosphorylation sites is of interest due to their importance in protein regulation; however, the identification of the exact sites of this modification is not always easily obtained due to the dynamic nature of phosphorylation and the challenges faced during mass spectrometric analysis. Herein we elaborate on our previous communication (Diedrich, J. K.; Julian, R. R. J. Am. Chem. Soc. 2008, 130, 12212-12213) describing a novel technique for assignment of phosphorylation in a site-specific and facile manner. Phosphorylation sites are selectively modified through β elimination followed by Michael addition chemistry to install a photolabile group. Photodissociation with 266 nm light yields homolytic cleavage at the modification site, generating a β radical which is poised to fragment the peptide backbone. Dissociation primarily yields d-type ions at the previously phosphorylated residue, allowing facile identification. Radical directed fragmentation also occurs in smaller abundances at neighboring residues. The mechanisms behind this selective radical fragmentation are presented and the utility is discussed. Fragmentation is shown to be independent of charge state allowing analysis of a wide variety of peptide sequences including peptides with multiple phosphorylation sites. A comparison of this technique is made with collision induced dissociation (CID) and electron capture dissociation (ECD) for representative peptides.
由于磷酸化在蛋白质调节中的重要性，磷酸化位点的鉴定备受关注；然而，由于磷酸化的动态性质以及在质谱分析中面临的挑战，这种修饰的确切位点的鉴定并不总是容易获得的。在此，我们详细阐述了我们之前的通讯（Diedrich，J. K.；Julian，R. R. J. Am. Chem. Soc. 2008，130，12212-12213）中描述的一种新颖技术，用于以特定和简便的方式分配磷酸化。通过β消除，随后进行迈克尔加成化学，选择性地修饰磷酸化位点以安装光不稳定基团。用 266nm 光进行光解，在修饰位点发生均裂裂解，生成β自由基，该自由基准备断裂肽骨架。主要在先前磷酸化的残基处生成 d 型离子，从而易于鉴定。在相邻残基处也以较小的丰度发生自由基定向断裂。提出了这种选择性自由基断裂的机制，并讨论了其用途。该技术的片段化独立于电荷状态，允许分析包括具有多个磷酸化位点的肽在内的各种肽序列。对代表性肽进行了与碰撞诱导解离（CID）和电子俘获解离（ECD）的比较。
