Ion Mobility-Mass Spectrometry Reveals Conformational Changes in Charge Reduced Multiprotein Complexes

Abstract

Characterizing intact multiprotein complexes in terms of both their mass and size by ion mobilitymass spectrometry is becoming an increasingly important tool for structural biology. Furthermore, the charge states of intact protein complexes can dramatically influence the information content of gas-phase measurements performed. Specifically, protein complex charge state has a demonstrated influence upon the conformation, mass resolution, ion mobility resolution, and dissociation properties of protein assemblies upon collisional activation. Here we present the first comparison of charge-reduced multiprotein complexes generated by solution additives and gas-phase ion-neutral reaction chemistry. While the charge reduction mechanism for both methods is undoubtedly similar, significant gas-phase activation of the complex is required to reduce the charge of the assemblies generated using the solution additive strategy employed here. This activation step can act to unfold intact protein complexes, making the data difficult to correlate with solution-phase structures and topologies. We use ion mobility-mass spectrometry to chart such conformational effects for a range of multi-protein complexes, and demonstrate that approaches to reduce charge based on ion-neutral reaction chemistry in the gas-phase consistently produce protein assemblies having compact, 'native-like' geometries while the same molecules added in solution generate significantly unfolded gas-phase complexes having identical charge states.

Conformational Space and Stability of ETD Charge Reduction Products of Ubiquitin

“…While charge reduction in the gas or droplet phase via ion/ion and ion/neutral chemistry has been performed before, the focus was on spectral simplification or deconvolution of overlapping signals in the m/z domain, rather than investigating the effect on gasphase ion structure [4,6,[25][26][27][28]. Efforts to combine this approach with ion mobility have mostly focused on large, noncovalent complexes, and in these cases, little or no conformational change has generally been observed [5,[29][30][31]. Our CCS values are larger than reported in a recent study [24]; however, that study used an IM calibration protocol [16] which relies on reference values measured in helium by the Clemmer group [14].…”

Extensive Charge Reduction and Dissociation of Intact Protein Complexes Following Electron Transfer on a Quadrupole-Ion Mobility-Time-of-Flight MS

“…Using this methodology, we are able to observe ions with a reduced net positive charge as low as 1+ from native protein complexes, whilst also generating product ions that yield sequence-specific information within the same experiment. Importantly, the ability to manipulate charge states in the gas phase over such a wide range also opens up new possibilities for the investigation of electrostatic effects on the folding state of protein ions in the gas phase, independent of how they were generated, and to minimize the effect of charge repulsion on native structures [33,34]. An interesting study in this regard was recently carried out by Campuzano and Schnier, who used a corona discharge to induce significant charge reduction of peptides and proteins on a Synapt HDMS instrument [35,36].…”

The Utilization of Chiral Ion Mobility Spectrometry for the Detection of Enantiomeric Mixtures and Thermally Labile Compounds

“…However, IMS has been able to not only detect these compounds, but do so with extreme efficiency. The 63 Ni-based IMS systems have been reported to detect these compounds in the part per billion to part per trillion detection range [17]. The low detection limits result from the seemingly tailor-made ionization mechanism scheme between the radioactive nickel ( 63 Ni) (see Equation 5) and many of these thermally labile species which contain electron receiving or electron withdrawing groups such as halides as nitrates.…”

“…The major drawback to using a traditional IMS system is that an interfering compound may be able to mask the presence of a target analyte from being detected. My project aims to alleviate this obstacle through the use of an ESI source rather than 63 Ni source. ESI, through the use of selected solvent systems, allows IMS to still be able to detect these thermally labile compounds but with the added advantage of ionizing most if not all analytes within a sample simultaneously.…”

“…[1;6]. The most common of these is radiation using a 10milli Currie 63 Ni source that emits beta particles with maximum energy of approximately 64KeV [1] and an average energy of 17KeV [7]. Other radioactive sources such as americium ( 241 Am) and tritium ( 3 H) provide ionization at different energy levels with 5.4MeV of alpha particles [6;43] and 18.6KeV of beta particle [43] respectively.…”