Ion Mobility Separation of Isomeric Phosphopeptides from a Protein with Variant Modification of Adjacent Residues

Ion mobility spectrometry (IMS), and particularly differential or field asymmetric waveform IMS (FAIMS), was recently shown capable of separating peptides with variant localization of post-translational modifications. However, that work was limited to a model peptide with Ser phosphorylation on fairly distant alternative sites. Here, we demonstrate that FAIMS (coupled to electrospray/mass spectrometry (ESI/MS)) can broadly baseline-resolve variant phosphopeptides from a biologically modified human protein, including those involving phosphorylation of different residues and adjacent sites that challenge existing tandem mass spectrometry (MS/MS) methods most. Singly and doubly phosphorylated variants can be resolved equally well and identified without dissociation, based on accurate separation properties. The spectra change little over a range of infusion solvent pH; hence, the present approach should be viable in conjunction with chromatographic separations using mobile phase gradients.