pFAIMS: An optimized workflow for quantitative multiplexed phosphorylation analysis using high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS)

Protein phosphorylation is vital for the regulation of cellular signaling. Isobaric tag-based proteomic techniques, such as tandem mass tags (TMT), can measure the relative phosphorylation states of peptides in a multiplexed format. However, the overall low stoichiometry of protein phosphorylation constrains the analytical depth of phosphopeptide analysis by mass spectrometry, thereby requiring robust and sensitive workflows. Here we evaluate and optimize high-Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) coupled to Orbitrap Tribrid mass spectrometers for the analysis of TMT10plex-labeled phosphopeptides. We determined that using FAIMS-SPS-MS3 with three compensation voltages (CV) in a single method minimizes inter-CV overlap and maximizes peptide coverage (e.g., CV=-40V/-60V/-80V) and that consecutive analyses using CID-MSA and HCD fragmentation at the MS2 stage increases the depth of phosphorylation analysis.