Fragmentation and Mobility Separation of Peptide and Protein Ions in a Trapped-Ion Mobility Device

Ion mobility separations (IMS) have increasingly been coupled with mass spectrometry to increase peak capacity and deconvolute complex mass spectra in proteomics workflows. IMS separations can be integrated prior to or following the collisional activation step. Post-activation IMS separations have demonstrated many advantages, yet few instrument platforms are capable of this feat. Here, we present the fragmentation of peptide ions within a commercially available trapped-ion mobility spectrometry device. Fragmentation is initiated prior to mobility analysis enabling the separation of generated product ions. The added separation step deconvolutes product ion spectra and permits improved annotation of product ions. Furthermore, we demonstrate the isolation and fragmentation of mobility separated product ions with the downstream quadrupole and collisional cell. When applied to melittin and ubiquitin, this ion mobility assisted pseudo-MS³ fragmentation approach generates sequence coverage ∼50% greater than that of typical MS² analyses. We envision this ion-mobility-assisted fragmentation technique as the foundation of a powerful new pseudo-MS³ workflow for application toward middle- or top-down proteomics.