Localization and Quantification of Post-Translational Modifications of Proteins Using Electron Activated Dissociation Fragmentation on a Fast-Acquisition Time-of-Flight Mass Spectrometer

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 MRMHR) 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 similar to 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.