Evaluation of a Bruker timsTOF Pro for Native Mass Spectrometry

Abstract

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.

Tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS): a promising analytical method for investigating heterogenous samples

“…7, which depicts cross-section distributions recorded for the small proteins ubiquitin 1,52 (8.6 kDa) and cytochrome c (12.4 kDa). 115 For charge state 6+ of ubiquitin, all spectra exhibit a single feature with cross sections in close agreement with the value reported by Bowers from a drift tube measurement (1200 Å 2 , Fig. 7A).…”

“…Hence, the guiding principle to maximizing the softness in TIMS is to minimize the force F acting on the analyte ion due to the applied electric fields and ion-ion interactions. 5,52,115 The second reason "softness" is critical is that the momentum transfer cross section of an ion (and thus its mobility) depends on the ion-neutral collision energy: 10,58 the cross section decreases non-linearly with effective ion temperature (mean collision energy). 33,113,[116][117][118][119][120][121] Hence, when ion mobility measurements are conducted under different effective temperatures, their measured cross sections differ even in the absence of any structural changes.…”

“…Hence, the guiding principle to maximizing the softness in TIMS is to minimize the force F acting on the analyte ion due to the applied electric fields and ion–ion interactions. 5,52,115…”

Expanding Orbitrap collision cross section measurements to native protein applications through kinetic energy and signal decay analysis

“…2,[15][16][17][18] These techniques have expanded the capabilities of IM-MS for the analysis of larger and more complex biomolecules. 19 Many commercial IM systems have CCS limits of approximately 4,000 Å 2 , 20 corresponding to protein mass of around 25-60 kDa depending on the charge state and whether the protein is native-like or denatured, but other home-built and commercial systems have approached the upper mass limit of the ionization sources used and the mass spectrometer itself. 21 For example, protein complexes up to 4 MDa and 800 kDa have been analyzed on drift tube 21 and traveling wave IM instruments, 22 respectively, and more recently home-built trapped ion mobility spectrometer approached an 800 kDa mass limit.…”

Top-Down Ion Mobility Separations of Isomeric Proteoforms

“…42 This shows the role of conformational annealing upon rf heating on a second timescale applicable in TIMS and FAIMS, but not DTIMS. While TIMS could be operated in a “soft” regime with minimal heating and conformational distortion of proteins, 43 that (unlike in native MS studies) is irrelevant here.…”