Characterizing the top-down sequencing of protein ions prior to mobility separation in a timsTOF

Mass spectrometry (MS)-based proteomics workflows of intact protein ions have increasingly been utilized to study biological systems. These workflows, however, frequently result in convoluted and difficult to analyze mass spectra. Ion mobility spectrometry (IMS) is a promising tool to overcome these limitations by separating ions by their mass- and size-to-charge ratios. In this work, we further characterize a newly developed method to collisionally dissociate intact protein ions in a trapped ion mobility spectrometry (TIMS) device. Dissociation occurs prior to ion mobility separation and thus, all product ions are distributed throughout the mobility dimension, enabling facile assignment of near isobaric product ions. We demonstrate that collisional activation within a TIMS device is capable of dissociating protein ions up to 66 kDa. We also demonstrate that the ion population size within the TIMS device significantly influences the efficiency of fragmentation. Lastly, we compare CIDtims to the other modes of collisional activation available on the Bruker timsTOF and demonstrate that the mobility resolution in CIDtims enables the annotation of overlapping fragment ions and improves sequence coverage.

Automated summary

Mass spectrometry-based proteomics workflows are widely used but often result in complex mass spectra. Ion mobility spectrometry (IMS) can separate ions based on their mass and size-to-charge ratios, providing a solution to this problem. This study characterizes a new method to dissociate intact protein ions in a trapped ion mobility spectrometry (TIMS) device, enabling easy identification of similar product ions. The results show that collisional activation within a TIMS device can dissociate protein ions up to 66 kDa and that the size of the ion population significantly affects fragmentation efficiency. Additionally, comparing collisional activation modes, CIDtims in TIMS improves annotation of overlapping fragment ions and enhances sequence coverage.