Nonlinear Frequency Modulation for Fourier Transform Ion Mobility Mass Spectrometry Improves Experimental Efficiency.

Through optimization of terminal frequencies and effective sampling rates, we have developed nonlinear sawtooth-shaped frequency sweeps for efficient Fourier transform ion mobility mass spectrometry (FT-IM-MS) experiments. This is in contrast to conventional FT-IM-MS experiments where ion gates are modulated according to a linear frequency sweep. Linear frequency sweeps are effective but can be hindered by the amount of useful signal obtained using a single sweep over a large frequency range imposed by ion gating inefficiencies, particularly small ion packets, and gate depletion. These negative factors are direct consequences of the inherently low gate pulse widths of high-frequency ion gating events, placing an upper bound on FT-IM-MS performance. Here, we report alternative ion modulation strategies. Sawtooth frequency sweeps may be constructed for the purpose of either extending high-SNR transients or conducting efficient signal-averaging experiments for low-SNR transients. The data obtained using this approach show high-SNR signals for a set of low-mass tetraalkylammonium salts (<1000 m/z) where resolving powers in excess of 500 are achieved. Data for low-SNR obtained for multimeric protein complexes streptavidin (53 kDa) and GroEL (800 kDa) also reveal large increases in the signal-to-noise ratio for reconstructed arrival time distributions.

Mid-infrared spectroscopy of 1-cyanonaphthalene cation for astrochemical consideration.

We present the low temperature gas-phase vibrational spectrum of ionised 1-cyanonaphthalene (1-CNN+) in the mid-infrared region. Experimentally, 1-CNN+ ions are cooled below 10 K in a cryogenic ion trapping apparatus, tagged with He atoms and probed with tuneable radiation. Quantum-chemical calculations are carried out at a density functional theory level. The spectrum is dominated by the CN-stretch at 4.516 µm, with weaker CH modes near 3.2 µm.

Modified Ion Source for the Improved Collisional Activation of Protein Complexes.

The analysis of large molecules is challenging, as they often have salts and adducts retained through the electrospray process, which increase the observed mass and compromise the achievable mass resolution. Mild collisional activation has been shown to be very effective for the removal of adducts and increases both measurement accuracy and mass resolution of large (>100 kDa) protein complexes. Collisionally activated protein ions are more completely desolvated due to the increased number of collisions when trapped following activation. A short square quadrupole maintained at 300 mTorr by a mechanical pump was added between the ion funnel and transmission quadrupole. This configuration and operation effectively removed adducts from the 800 kDa tetradecamer GroEL as well as fragmented smaller protein complexes like C-reactive protein. Due to the gas high pressure, ions of low size-to-charge ratio, such as those in charge reducing buffers, had low ejection efficiency. We show that segmenting the quadrupole rods greatly improves signal intensity for charge reduced GroEL D398A mutant compared to nonsegmented rods when operating at high pressure.

Optimization of a Digital Mass Filter for the Isolation of Intact Protein Complexes in Stability Zone 1,1.

Digital mass filters are advantageous for the analysis of large molecules due to the ability to perform ion isolation of high-m/z ions without the generation of very high radio frequency (RF) and DC voltages. Experimentally determined Mathieu stability diagrams of stability zone 1,1 for capacitively coupled digital waveforms show a voltage offset between the quadrupole rod pairs is introduced by the capacitors which is dependent on the voltage magnitude of the waveform and the duty cycle. This changes the ion's a value from a = 0 to a < 0. These effects are illustrated for isolation for single-charge states for various protein complexes up to 800 kDa (GroEL) for stability zone 1,1. Isolation resolving power (m/Δm) of approximately 280 was achieved for an ion of m/z 12,315 (z = 65+ for 800.5 kDa GroEL D398A), which corresponds to an m/z window of 44.

Surface Activity of Amines Provides Evidence for the Combined ESI Mechanism of Charge Reduction for Protein Complexes.

Charge reduction reactions are important for native mass spectrometry (nMS) because lower charge states help retain native-like conformations and preserve noncovalent interactions of protein complexes. While mechanisms of charge reduction reactions are not well understood, they are generally achieved through the addition of small molecules, such as polyamines, to traditional nMS buffers. Here, we present new evidence that surface-active, charge reducing reagents carry away excess charge from the droplet after being emitted due to Coulombic repulsion, thereby reducing the overall charge of the droplet. Furthermore, these processes are directly linked to two mechanisms for electrospray ionization, specifically the charge residue and ion evaporation models (CRM and IEM). Selected protein complexes were analyzed in solutions containing ammonium acetate and selected trialkylamines or diaminoalkanes of increasing alkyl chain lengths. Results show that amines with higher surface activity have increased propensities for promoting charge reduction of the protein ions. The electrospray ionization (ESI) emitter potential was also found to be a major contributing parameter to the prevalence of charge reduction; higher emitter potentials consistently coincided with lower average charge states among all protein complexes analyzed. These results offer experimental evidence for the mechanism of charge reduction in ESI and also provide insight into the final stages of the ESI and their impact on biological ions.

Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP), and Stoichiometry of GroEL-GroES Complexes.

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, a chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL-GroES complexes. The results show clear evidence for destabilization of both GroEL14 and GroES7 at temperatures of 50 and 45 °C, respectively, substantially below the previously reported melting temperature (Tm ∼ 70 °C). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESy-ATPn, where y = 1, 2, 8 and n = 0, 1, 2, 8, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to temperature effects: (i) temperature-dependent ATP binding to GroEL; (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 and 40 °C. The similarities between results obtained by using native MS and cryo-EM [Clare et al. An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-911; Ranson et al. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.Nat. Struct. Mol. Biol. 2006, 13, 147-152] underscore the utility of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperonin-assisted protein folding cycle.

Implementing Digital-Waveform Technology for Extended m/z Range Operation on a Native Dual-Quadrupole FT-IM-Orbitrap Mass Spectrometer.

Here, we describe a digital-waveform dual-quadrupole mass spectrometer that enhances the performance of our drift tube FT-IMS high-resolution Orbitrap mass spectrometer (MS). The dual-quadrupole analyzer enhances the instrument capabilities for studies of large protein and protein complexes. The first quadrupole (q) provides a means for performing low-energy collisional activation of ions to reduce or eliminate noncovalent adducts, viz., salts, buffers, detergents, and/or endogenous ligands. The second quadrupole (Q) is used to mass-select ions of interest for further interrogation by ion mobility spectrometry and/or collision-induced dissociation (CID). Q is operated using digital-waveform technology (DWT) to improve the mass selection compared to that achieved using traditional sinusoidal waveforms at floated DC potentials (>500 V DC). DWT allows for increased precision of the waveform for a fraction of the cost of conventional RF drivers and with readily programmable operation and precision (Hoffman, N. M. . A comparison-based digital-waveform generator for high-resolution duty cycle. Review of Scientific Instruments 2018, 89, 084101).

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding.

Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated. Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from ∼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold- and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.

THE IMS PARADOX: A PERSPECTIVE ON STRUCTURAL ION MOBILITY-MASS SPECTROMETRY.

Studies of large proteins, protein complexes, and membrane protein complexes pose new challenges, most notably the need for increased ion mobility (IM) and mass spectrometry (MS) resolution. This review covers evolutionary developments in IM-MS in the authors' and key collaborators' laboratories with specific focus on developments that enhance the utility of IM-MS for structural analysis. IM-MS measurements are performed on gas phase ions, thus "structural IM-MS" appears paradoxical-do gas phase ions retain their solution phase structure? There is growing evidence to support the notion that solution phase structure(s) can be retained by the gas phase ions. It should not go unnoticed that we use "structures" in this statement because an important feature of IM-MS is the ability to deal with conformationally heterogeneous systems, thus providing a direct measure of conformational entropy. The extension of this work to large proteins and protein complexes has motivated our development of Fourier-transform IM-MS instruments, a strategy first described by Hill and coworkers in 1985 (Anal Chem, 1985, 57, pp. 402-406) that has proved to be a game-changer in our quest to merge drift tube (DT) and ion mobility and the high mass resolution orbitrap MS instruments. DT-IMS is the only method that allows first-principles determinations of rotationally averaged collision cross sections (CSS), which is essential for studies of biomolecules where the conformational diversities of the molecule precludes the use of CCS calibration approaches. The Fourier transform-IM-orbitrap instrument described here also incorporates the full suite of native MS/IM-MS capabilities that are currently employed in the most advanced native MS/IM-MS instruments. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

First-Principles Collision Cross Section Measurements of Large Proteins and Protein Complexes.

Rotationally averaged collision cross section (CCS) values for a series of proteins and protein complexes ranging in size from 8.6 to 810 kDa are reported. The CCSs were obtained using a native electrospray ionization drift tube ion mobility-Orbitrap mass spectrometer specifically designed to enhance sensitivity while having high-resolution ion mobility and mass capabilities. Periodic focusing (PF)-drift tube (DT)-ion mobility (IM) provides first-principles determination of the CCS of large biomolecules that can then be used as CCS calibrants. The experimental, first-principles CCS values are compared to previously reported experimentally determined and computationally calculated CCS using projected superposition approximation (PSA), the Ion Mobility Projection Approximation Calculation Tool (IMPACT), and Collidoscope. Experimental CCS values are generally in agreement with previously reported CCSs, with values falling within ∼5.5%. In addition, an ion mobility resolution (CCS centroid divided by CCS fwhm) of ∼60 is obtained for pyruvate kinase (MW ∼ 233 kDa); however, ion mobility resolution for bovine serum albumin (MW ∼ 68 kDa) is less than ∼20, which arises from sample impurities and underscores the importance of sample quality. The high resolution afforded by the ion mobility-Orbitrap mass analyzer provides new opportunities to understand the intricate details of protein complexes such as the impact of post-translational modifications (PTMs), stoichiometry, and conformational changes induced by ligand binding.