Surface-Induced Dissociation of Protein Complexes Selected by Trapped Ion Mobility Spectrometry.

Native mass spectrometry (nMS), particularly in conjunction with gas-phase ion mobility spectrometry measurements, has proven useful as a structural biology tool for evaluating the stoichiometry, conformation, and topology of protein complexes. Here, we demonstrate the combination of trapped ion mobility spectrometry (TIMS) and surface-induced dissociation (SID) on a Bruker SolariX XR 15 T FT-ICR mass spectrometer for the structural analysis of protein complexes. We successfully performed SID on mobility-selected protein complexes, including the streptavidin tetramer and cholera toxin B with bound ligands. Additionally, TIMS-SID was employed on a mixture of the peptides desArg1 and desArg9 bradykinin to mobility-separate and identify the individual peptides. Importantly, results show that native-like conformations can be maintained throughout the TIMS analysis. The TIMS-SID spectra are analogous to SID spectra acquired using quadrupole mass selection, indicating little measurable, if any, structural rearrangement during mobility selection. Mobility parking was used on the ion or mobility of interest and 50-200 SID mass spectra were averaged. High-quality TIMS-SID spectra were acquired over a period of 2-10 min, comparable to or slightly longer than SID coupled with ion mobility on various instrument platforms in our laboratory. The ultrahigh resolving power of the 15 T FT-ICR allowed for the identification and relative quantification of overlapping SID fragments with the same nominal m/z based on isotope patterns, and it shows promise as a platform to probe small mass differences, such as protein/ligand binding or post-translational modifications. These results represent the potential of TIMS-SID-MS for the analysis of both protein complexes and peptides.

Simple and Minimally Invasive SID Devices for Native Mass Spectrometry.

We describe a set of simple devices for surface-induced dissociation of proteins and protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few millimeters along the ion path axis) and is easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. Despite the decrease in size and reduction in number of electrodes to 3 (from 10 to 12 in Gen 1 and ∼6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.

Ion Mobility and Surface Collisions: Submicrometer Capillaries Can Produce Native-like Protein Complexes.

The use of submicrometer capillaries for nanoelectrospray ionization of native proteins and protein complexes effectively reduces the number of nonspecific salt adducts to biological molecules, therefore increasing the apparent resolution of a mass spectrometer without any further instrument modifications or increased ion activation. However, the increased interaction between proteins and the surface of the capillary has been shown to promote protein expansion and therefore loss of native structure. Here, we compare the effect of micrometer and submicrometer sized capillaries on the native structures of the protein complexes streptavidin, concanavalin A, and C-reactive protein under charge reducing conditions. We observe that the use of submicrometer capillaries did not result in a significantly higher charge state distribution, indicative of expansion, when compared to micrometer sized capillaries for complexes in 100 mM ammonium acetate and 100 mM triethylammonium acetate and for streptavidin in 200 mM ammonium acetate with no charge reduction. Additionally, no significant differences in collision cross sections were observed using ion mobility mass spectrometry. Finally, the dissociation behaviors of protein complexes ionized using micrometer and submicrometer capillaries were compared to determine if any structural perturbation occurred during ionization. Protein complexes from both capillary sizes displayed similar surface-induced dissociation patterns at similar activation energies. The results suggest that submicrometer capillaries do not result in significant changes to protein complex structure under charge reducing conditions and may be used for native mass spectrometry experiments. Submicrometer capillaries can be used to resolve small mass differences of biological systems on a QTOF platform; however, a laser tip puller is required for pulling reproducible submicrometer capillaries, and disruption in spray due to clogging was observed for larger protein complexes.

Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Top-Down Protein Analysis by Tandem-Trapped Ion Mobility Spectrometry/Mass Spectrometry (Tandem-TIMS/MS) Coupled with Ultraviolet Photodissociation (UVPD) and Parallel Accumulation/Serial Fragmentation (PASEF) MS/MS Analysis.

"Top-down" proteomics analyzes intact proteins and identifies proteoforms by their intact mass as well as the observed fragmentation pattern in tandem mass spectrometry (MS/MS) experiments. Recently, hybrid ion mobility spectrometry-mass spectrometry (IM/MS) methods have gained traction for top-down experiments, either by allowing top-down analysis of individual isomers or alternatively by improving signal/noise and dynamic range for fragment ion assignment. We recently described the construction of a tandem-trapped ion mobility spectrometer/mass spectrometer (tandem-TIMS/MS) coupled with an ultraviolet (UV) laser and demonstrated a proof-of-principle for top-down analysis by UV photodissociation (UVPD) at 2-3 mbar. The present work builds on this with an exploration of a top-down method that couples tandem-TIMS/MS with UVPD and parallel-accumulation serial fragmentation (PASEF) MS/MS analysis. We first survey types and structures of UVPD-specific fragment ions generated in the 2-3 mbar pressure regime of our instrument. Notably, we observe UVPD-induced fragment ions with multiple conformations that differ from those produced in the absence of UV irradiation. Subsequently, we discuss how MS/MS spectra of top-down fragment ions lend themselves ideally for probability-based scoring methods developed in the bottom-up proteomics field and how the ability to record automated PASEF-MS/MS spectra resolves ambiguities in the assignment of top-down fragment ions. Finally, we describe the coupling of tandem-TIMS/MS workflows with UVPD and PASEF-MS/MS analysis for native top-down protein analysis.