FAIMS enhances the detection of PTM crosstalk sites

Protein post-translational modification (PTMs) enable cells to rapidly change in response to biological stimuli. With over 400 PTMs, understanding these control mechanisms is complex. To date, efforts have focused on investigating the effect of single PTMs on protein function. Yet, many proteins contain multiple PTMs. Moreover, one PTM can alter the prevalence of another; a phenomenon termed PTM crosstalk. Understanding PTM crosstalk is critical; however, its detection is challenging since PTMs occur sub-stoichiometrically. In this work, we developed an enrichment-free, label-free proteomics method that utilized high field asymmetric ion mobility spectrometry (FAIMS) to enhance the detection of PTM crosstalk. We showed that by searching for multiple combinations of dynamic PTMs on peptide sequences, a large increase in identifications of candidate PTM crosstalk sites was observed compared with standard LC-MS/MS. Additionally, by cycling through FAIMS compensation voltages within a single LC-FAIMS-MS/MS run, we showed that our LC-FAIMS-MS/MS workflow could increase multi-PTM containing peptide identifications without additional increases in run times. With more novel candidate crosstalk sites identified, we envisage LC-FAIMS-MS/MS to play an important role in expanding the repertoire of multi-PTM identifications. Moreover, it is only by detecting PTM crosstalk, that we can ‘see’ the full picture of how proteins are regulated.