Buck Institute for Research on Aging, Novato, California 94947, United States.
SCIEX, Redwood City, California 94065, United States.
J Am Soc Mass Spectrom. 2023 Oct 4;34(10):2199-2210. doi: 10.1021/jasms.3c00144. Epub 2023 Sep 11.
Protein post-translational modifications (PTMs) are crucial and dynamic players in a large variety of cellular processes and signaling. Proteomic technologies have emerged as the method of choice to profile PTMs. However, these analyses remain challenging due to potential low PTM stoichiometry, the presence of multiple PTMs per proteolytic peptide, PTM site localization of isobaric peptides, and neutral losses. Collision-induced dissociation (CID) is commonly used to characterize PTMs, but the application of collision energy can lead to neutral losses and incomplete peptide sequencing for labile PTM groups. In this study, we assessed the performance of an alternative fragmentation, electron activated dissociation (EAD), to characterize, site localize, and quantify peptides with labile modifications in comparison to CID, both operated on a recently introduced fast-scanning quadrupole-time-of-flight (QqTOF) mass spectrometer. We analyzed biologically relevant phosphorylated, succinylated, malonylated, and acetylated synthetic peptides using targeted parallel reaction monitoring (PRM or MRM) assays. We report that electron-based fragmentation preserves the malonyl group from neutral losses. The novel tunable EAD kinetic energy maintained labile modification integrity and provided better peptide sequence coverage with strong PTM-site localization fragment ions. Activation of a novel trap-and-release technology significantly improves the duty cycle and provided significant MS/MS sensitivity gains by an average of 6-11-fold for EAD analyses. Evaluation of the quantitative EAD PRM workflows revealed high reproducibility with coefficients of variation of ∼2-7%, as well as very good linearity and quantification accuracy. This novel workflow combining EAD and trap-and-release technology provides high sensitivity, alternative fragmentation information to achieve confident PTM characterization and quantification.
蛋白质翻译后修饰（PTMs）是多种细胞过程和信号转导中至关重要且动态的参与者。蛋白质组学技术已成为描绘 PTMs 的首选方法。然而，由于潜在的低 PTM 化学计量、每个蛋白水解肽中存在多种 PTM、等电点肽的 PTM 位点定位和中性损失，这些分析仍然具有挑战性。碰撞诱导解离（CID）通常用于表征 PTMs，但碰撞能的应用可能导致不稳定 PTM 基团的中性损失和不完全肽测序。在这项研究中，我们评估了替代碎裂方法——电子激活解离（EAD）的性能，以与 CID 相比，对具有不稳定修饰的肽进行特征分析、定位和定量，这两种方法都在最近引入的快速扫描四极杆-飞行时间（QqTOF）质谱仪上进行。我们使用靶向平行反应监测（PRM 或 MRM）测定法分析了具有生物学意义的磷酸化、琥珀酰化、丙二酰化和乙酰化合成肽。我们报告说，基于电子的碎裂可防止丙二酰基发生中性损失。新型可调谐 EAD 动能可保持不稳定修饰的完整性，并提供更好的肽序列覆盖，具有强烈的 PTM 位点定位片段离子。新型陷阱和释放技术的激活可显著提高工作周期，并通过平均 6-11 倍的 EAD 分析获得显著的 MS/MS 灵敏度增益。对定量 EAD PRM 工作流程的评估显示，具有约 2-7%的变异系数的高重复性，以及非常好的线性度和定量准确性。这种结合 EAD 和陷阱和释放技术的新型工作流程提供了高灵敏度和替代的碎裂信息，可实现可靠的 PTM 特征和定量。
