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 ∼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.