Differentiation of Repeat Phosphopeptide Isomers Through Metal Interactions in FAIMS

Phosphorylation regulates RNA polymerase II (RNAPII) to transcribe RNA from DNA but remains challenging to study by standard liquid chromatography followed by electrospray ionized-mass spectrometry (LC/ESI-MS) and fragmentation studies. To this end, we employed field asymmetric waveform ion mobility spectrometry (FAIMS) to explore metal-phosphorylation adduction to facilitate the analysis of variants. We analyzed phosphoisomers inspired by RNAPII C-terminal domain (CTD) heptads YSPTSPS, indistinguishable by collision-induced dissociation. The modulation of the carrier gas with solvent modifier provided partial differentiation of gas-phase metal-peptide adducts. During the compensation voltage scan with increasing dispersion voltage, alkali metal-doped phosphoheptad isomers showed selective elution. As per PM7 models, isomers may cluster/decluster with alkali ion/methanol with different bond energies due to the varied phosphosites. These conditions were successfully applicable to biologically significant modified tryptic diheptads. Translation of such methods might allow the detection of phosphosites in finding disease-causing mutations or aid targeted therapeutics development.