Challenges and Opportunities to Quantify Post-Translational Modifications Using Novel Proteomic Workflows and Approaches

The dynamic nature of post-translational modifications (PTMs) enables instantaneous intracellular signaling events. We are interested in a diverse range of modifications such protein acylation in liver and brown adipose fat tissues, malonylation and succinylation in the brain during aging and disease, succinylation during kidney injury, as well as phosphorylation in other biological systems. We have been using fast scanning TripleTOF systems to analyze a variety of PTMs using both data independent acquisitions (DIA) and more recently multiple reaction monitoring-high resolution (MRMHR) to identify, characterize, and quantify PTM-containing peptides in a high-throughput format. While rich information has been obtained using traditional CID fragmentation approaches, many of the modifications we study are highly labile and difficult to characterize. Hence we evaluated a new ion fragmentation approach for its impact on improved PTM characterization and more detailed site localization. Using examples of key modifications found in our sirtuin 5 mouse models we will demonstrate the impact of this new fragmentation approach on assessing the PTM profile during kidney injury, and potential pathways to protect from kidney injury via elevated succinylation levels which could open therapeutic avenues.