Mass-spectrometry-based methods have made significant progress in the characterization of post-translational modifications (PTMs) in peptides and proteins; however, room remains to improve fragmentation methods. Ideal MS/MS methods are expected to simultaneously provide extensive sequence information and localization of PTM sites and retain labile PTM groups. This collection of criteria is difficult to meet, and the various activation methods available today offer different capabilities. In order to examine the specific case of phosphorylation on peptides, we investigate electron transfer dissociation (ETD), electron-activated dissociation (EAD), and 193 nm ultraviolet photodissociation (UVPD) and compare all three methods with classical collision-induced dissociation (CID). EAD and UVPD show extensive backbone fragmentation, comparable in scope to that of CID. These methods provide diverse backbone fragmentation, producing a/x, b/y, and c/z ions with substantial sequence coverages. EAD displays a high retention efficiency of the phosphate modification, attributed to its electron-mediated fragmentation mechanisms, as observed in ETD. UVPD offers reasonable retention efficiency, also allowing localization of the PTM site. EAD experiments were also performed in an LC-MS/MS workflow by analyzing phosphopeptides spiked in human plasma, and spectra allow accurate identification of the modified sites and discrimination of isomers. Based on the overall performance, EAD and 193 nm UVPD offer alternative options to CID and ETD for phosphoproteomics.

中文翻译：

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通过电子激活和紫外解离质谱表征磷酸化肽：与碰撞诱导解离的比较研究

基于质谱的方法在肽和蛋白质翻译后修饰 (PTM) 的表征方面取得了重大进展；然而，分段方法仍有改进的空间。理想的 MS/MS 方法预计能够同时提供广泛的序列信息和 PTM 位点定位并保留不稳定的 PTM 基团。这一系列标准很难满足，并且当今可用的各种激活方法提供了不同的功能。为了检查肽磷酸化的具体情况，我们研究了电子转移解离 (ETD)、电子激活解离 (EAD) 和 193 nm 紫外光解离 (UVPD)，并将所有三种方法与经典碰撞诱导解离 (CID) 进行比较）。 EAD 和 UVPD 显示出广泛的主链断裂，其范围与 CID 相当。这些方法提供了不同的主链断裂，产生具有大量序列覆盖的a / x、b / y和c / z离子。正如 ETD 中所观察到的，EAD 对磷酸盐修饰表现出较高的保留效率，这归因于其电子介导的断裂机制。 UVPD 提供合理的保留效率，还允许 PTM 位点的本地化。 EAD 实验还在 LC-MS/MS 工作流程中进行，通过分析人血浆中掺入的磷酸肽，光谱可以准确识别修饰位点并区分异构体。根据整体性能，EAD 和 193 nm UVPD 为磷酸蛋白质组学提供了 CID 和 ETD 的替代选择。