Abstract
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.