Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their abil-ity to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capa-ble of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that continues to grow in utilization due to its inherently high resolution and re-duced instrumental footprint. In currently deployed instruments, however, typical modes of collisional activation do not precede IMS analysis and thus, the instruments are incapable of performing CIU workflows. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolu-tion provided by the TIMS separation uncovers previously obscured unfolding complexity.