Evaluation of a Bruker timsTOF Pro for Native Mass Spectrometry

Mass spectrometry-based assays in structural biology studies typically measure either intact or digested proteins. Traditionally, there are different mass spectrometers dedicated for such measurements: those focused on the rapid analysis of small molecules, such as digested peptides, and those designed for high mass intact analysis. The Bruker timsTOF Pro mass spectrometer (ion mobility-quadrupole-time of flight platform, IM-Q-TOF), has become widely utilized for the analysis of small molecules in the fields of proteomics and metabolomics, with ion mobility spectrometry offering an additional stage of ion separation coupled to liquid chromatography. While this instrument has proven capabilities for small molecule analysis, the ability to perform high-quality native mass spectrometry of intact protein complexes remains largely uninvestigated. Here, we evaluate this IM-Q-TOF platform for the analysis of intact proteins and non-covalently bound protein complexes as small as 12 kDa (cytochrome c) and as large as 801 kDa (GroEL), utilizing the full range of ion mobility, MS, and MS/MS (in-source cleanup and collision cell CID) experiments available on this platform. In-source activation and collision cell CID were found to be robust capabilities for both small and large complexes. Nonetheless, the TIMS analyzer was soft enough to preserve protein-ligand interactions between 1,3-benzenedisulfonamide and carbonic anhydrase. TIMS-CID was performed on the protein complexes streptavidin (53 kDa), avidin (68 kDa) and cholera toxin B (CTB, 58 kDa). Pyruvate kinase and GroEL were beyond the trapping capabilities of the commercial TIMS analyzer. Although quadrupole selection is limited to m/z 3000 by the manufacturer, ions of significantly higher m/z can be transmitted and studied. The present results show that the commercially available Bruker IM-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for the TIMS analyzer and quadrupole will be required if protein complexes greater than a few tens of kDa are of interest.