Joshua A. Silveira

Fundamentals of Trapped Ion Mobility Spectrometry

AbstractTrapped ion mobility spectrometry (TIMS) is a relatively new gas-phase separation method that has been coupled to quadrupole orthogonal acceleration time-of-flight mass spectrometry. The TIMS analyzer is a segmented rf ion guide wherein ions are mobility-analyzed using an electric field that holds ions stationary against a moving gas, unlike conventional drift tube ion mobility spectrometry where the gas is stationary. Ions are initially trapped, and subsequently eluted from the TIMS analyzer over time according to their mobility (K). Though TIMS has achieved a high level of performance (R > 250) in a small device (<5 cm) using modest operating potentials (<300 V), a proper theory has yet to be produced. Here, we develop a quantitative theory for TIMS via mathematical derivation and simulations. A one-dimensional analytical model, used to predict the transit time and theoretical resolving power, is described. Theoretical trends are in agreement with experimental measurements performed as a function of K, pressure, and the axial electric field scan rate. The linear dependence of the transit time with 1/K provides a fundamental basis for determination of reduced mobility or collision cross section values by calibration. The quantitative description of TIMS provides an operational understanding of the analyzer, outlines the current performance capabilities, and provides insight into future avenues for improvement. Graphical Abstractᅟ

High Resolution Trapped Ion Mobility Spectrometery of Peptides

In the present work, we employ trapped ion mobility spectrometry (TIMS) for conformational analysis of several model peptides. The TIMS distributions are extensively compared to recent ion mobility spectrometry (IMS) studies reported in the literature. At a resolving power (R) exceeding 250, many new features, otherwise hidden by lower resolution IMS analyzers, are revealed. Though still principally limited by the plurality of conformational states, at present, TIMS offers R up to ∼3 to 8 times greater than modern drift tube or traveling wave IMS techniques, respectively. Unlike differential IMS, TIMS not only is able to resolve congested conformational features but also can be used to determine information about their relative size, via the ion-neutral collision cross section, offering a powerful new platform to probe the structure and dynamics of biochemical systems in the gas phase.

Cryogenic Ion Mobility-Mass Spectrometry Captures Hydrated Ions Produced During Electrospray Ionization

Evaporation of water from extensively hydrated protons and peptides formed by electrospray ionization (ESI) has been examined for the first time by cryogenic ion mobility-mass spectrometry (IM-MS). The extent of hydration was controlled using a heated capillary inlet operated between 340 and 391 K. Cold cluster ions formed in the source region were transported into a low temperature (∼80 K) IM drift tube using an electrostatic ion guide where they were separated on the basis of size-to-charge via low-energy collisions with helium gas. The eluting IM profile was subsequently pulsed into an orthogonal time-of-flight (TOF) mass spectrometer for mass-to-charge (m/z) identification of the cluster ion species. Key parameters that influence the cluster distributions were critically examined including the inlet temperature, drift tube temperature, and IM field strength. In agreement with previous studies, our findings indicate that water evaporation is largely dependent upon the particular charge-carrying species within the cluster. IM-MS results for protonated water clusters suggest that the special stability of H(+)(H(2)O)(n) (n = 21) is attributed to the presence of a compact isomer (assigned to a clathrate cage) that falls below the trendline produced by adjacent clusters in the n = 15 to 35 size range. Peptide studies are also presented in which specific and nonspecific solvation is observed for gramicidin S [GS + 2H](2+)(H(2)O)(n) (n = 0 to ∼26) and bradykinin [BK + 2H](2+)(H(2)O)(n) (n = 0 to ∼73), respectively.

Evolution of Hydrogen-Bond Networks in Protonated Water Clusters H(+)(H2O)n (n = 1 to 120) Studied by Cryogenic Ion Mobility-Mass Spectrometry.

Cryogenic (80 K) ion mobility-mass spectrometry (cryo-IM-MS) is employed to study structural transitions of protonated water clusters in both the small, H(+)(H2O)n (n = 1 to 30), and large, (n = 31 to ∼120), size regions. In agreement with previous studies, we find compelling evidence of regions of uniform cluster decay in the small size region, accompanied by sharp transition points whereby the loss of a single water monomer induces a different H-bonding motif. The investigation of the isomeric distribution of each species at 80 K reveals experimental evidence supporting the notion that H(+)(H2O)n (n = 6) is the smallest system to possess both Eigen- (H3O(+)) and Zundel- (H5O2(+)) centered structures. Cryo-IM-MS is particularly well-suited for studying clusters in the large size region, for which previous spectroscopic experimental studies are scarce.

From Solution to the Gas Phase: Factors That Influence Kinetic Trapping of Substance P in the Gas Phase

Substance P (RPKPQQFFGLM-NH2) [M + 3H](3+) ions have been shown to exist as two conformers: one that is kinetically trapped and one that is thermodynamically more stable and therefore energetically preferred. Molecular dynamics (MD) simulations suggested that the kinetically trapped population is stabilized by interactions between the charge sites and the polar side chains of glutamine (Q) located at positions 5 and 6 and phenylalanine (F) located at positions 7 and 8. Here, the individual contributions of these specific intramolecular interactions are systematically probed through site-directed alanine mutations of the native amino acid sequence. Ion mobility spectrometry data for the mutant peptide ions confirm that interactions between the charge sites and glutamine/phenylalanine (Q/F) side chains afford stabilization of the kinetically trapped ion population. In addition, experimental data for proline-to-alanine mutations at positions 2 and 4 clearly show that interactions involving the charge sites and the Q/F side chains are altered by the cis/trans orientations of the proline residues and that mutation of glycine to proline at position 9 supports results from MD simulations suggesting that the C-terminus also provides stabilization of the kinetically trapped conformation.

Damping factor links periodic focusing and uniform field ion mobility for accurate determination of collision cross sections.

The methodology for obtaining accurate ion-neutral collision cross section (Ω) values for peptides and proteins using periodic focusing ion mobility spectrometry (PF IMS) is presented. A mobility dampening factor (represented by the term α) is introduced to account for the relative increase in ion-neutral collisions in PF IMS compared to uniform field ion mobility spectrometry (UF IMS) for equivalent operating conditions. The results show that α may be easily quantified both theoretically and empirically for a specific PF IMS design operating at a given pressure based upon the charge state of the analyte. By simply incorporating an α term into traditional UF IMS expressions, accurate Ω values were obtained with excellent agreement (≤4% difference) compared to UF IMS measurements found in the current literature.

Unfolding of Hydrated Alkyl Diammonium Cations Revealed by Cryogenic Ion Mobility-Mass Spectrometry.

Hydration of the ammonium ion plays a key role in determining the biomolecular structure as well as local structure of water in aqueous environments. Experimental data obtained by cryogenic ion mobility-mass spectrometry (cryo-IM-MS) show that dehydration of alkyl diammonium cations induces a distinct unfolding transition at a critical number of water molecules, n = 21 to 23, n = 24 to 26, and n = 27 to 29, for 1,7-diaminoheptane, 1,8-diaminooctane, and 1,10-diaminodecane, respectively. Results are also presented that reveal compelling evidence for unique structural transitions of hydrated ammonium ions associated with the development of the hydrogen-bond network around individual charged groups. The ability to track the evolution of structure upon stepwise dehydration provides direct insight into the intricate interplay between solvent-molecule interactions that are responsible for defining conformations. Such insights are potentially valuable in understanding how ammonium ion solvation influences conformation(s) of larger biomolecules.

From Solution to Gas Phase: The Implications of Intramolecular Interactions on the Evaporative Dynamics of Substance P During Electrospray Ionization

Substance P (RPKPQQFFGLM-NH2) [M + 3H](3+) ions have been shown to occupy two distinct conformer states, a compact population of conformers that is formed by evaporation of hydrated ions, and an elongated population of conformers that is formed by collisional heating of the compact conformer. Molecular dynamics (MD) simulations and amino acid mutations revealed that the compact conformer is stabilized by intramolecular interactions between the localized charge-carrying sites, specifically the N-terminus, R(1), and K(3), with the side chains of glutamine and phenylalanine residues present in the peptide. Here, we employ amino acid mutations and cryogenic ion mobility-mass spectrometry (cryo-IM-MS) in an effort to understand how eliminating specific intramolecular interactions alters ion hydration, as well as the dehydration dynamics of substance P during the final stages of the electrospray process. The results clearly illustrate a direct link between the stabilizing effects of intramolecular self-solvation and the formation of substance P [M + 3H](3+) ions. Most notably, removal of these stabilizing interactions leads to a reduction in the abundances of [M + 3H](3+) ions induced by charge reduction reactions, i.e., loss of H(+)(H2O)n ions to form [M + 2H](2+) ions during the final stages of the electrospray process.

The Periodic Focusing Ion Funnel: Theory, Design, and Experimental Characterization by High-Resolution Ion Mobility-Mass Spectrometry

Simulation-based development and experimental characterization of a DC-only ion funnel is described herein. Radial ion confinement is achieved via periodic focusing whereby a collisionally dampened effective potential is generated in the inertial frame of an ion traversing the device with appreciable velocity. The new device, termed a periodic focusing ion funnel (PF IF), provides an efficient alternative to the rf ion funnel providing high ion transmission with fewer electrodes, simplified electrical circuitry, and reduced power supply requirements. The utility of the PF IF for structural ion mobility-mass spectrometry (IM-MS) studies is demonstrated using model peptide ions (bradykinin, gramicidin S, and trpzip 1).

Cryogenic Ion Mobility-Mass Spectrometry: Tracking Ion Structure from Solution to the Gas Phase.

Electrospray ionization (ESI) combined with ion mobility-mass spectrometry (IM-MS) is adding new dimensions, that is, structure and dynamics, to the field of biological mass spectrometry. There is increasing evidence that gas-phase ions produced by ESI can closely resemble their solution-phase structures, but correlating these structures can be complicated owing to the number of competing effects contributing to structural preferences, including both inter- and intramolecular interactions. Ions encounter unique hydration environments during the transition from solution to the gas phase that will likely affect their structure(s), but many of these structural changes will go undetected because ESI-IM-MS analysis is typically performed on solvent-free ions. Cryogenic ion mobility-mass spectrometry (cryo-IM-MS) takes advantage of the freeze-drying capabilities of ESI and a cryogenically cooled IM drift cell (80 K) to preserve extensively solvated ions of the type [M + xH](x+)(H2O)n, where n can vary from zero to several hundred. This affords an experimental approach for tracking the structural evolution of hydrated biomolecules en route to forming solvent-free gas-phase ions. The studies highlighted in this Account illustrate the varying extent to which dehydration can alter ion structure and the overall impact of cryo-IM-MS on structural studies of hydrated biomolecules. Studies of small ions, including protonated water clusters and alkyl diammonium cations, reveal structural transitions associated with the development of the H-bond network of water molecules surrounding the charge carrier(s). For peptide ions, results show that water networks are highly dependent on the charge-carrying species within the cluster. Specifically, hydrated peptide ions containing lysine display specific hydration behavior around the ammonium ion, that is, magic number clusters with enhanced stability, whereas peptides containing arginine do not display specific hydration around the guanidinium ion. Studies on the neuropeptide substance P illustrate the ability of cryo-IM-MS to elucidate information about heterogeneous ion populations. Results show that a kinetically trapped conformer is stabilized by a combination of hydration and specific intramolecular interactions, but upon dehydration, this conformer rearranges to form a thermodynamically favored gas-phase ion conformation. Finally, recent studies on hydration of the protein ubiquitin reveal water-mediated dimerization, thereby illustrating the extension of this approach to studies of large biomolecules. Collectively, these studies illustrate a new dimension to studies of biomolecules, resulting from the ability to monitor snapshots of the structural evolution of ions during the transition from solution to gas phase and provide unparalleled insights into the intricate interplay between competing effects that dictate conformational preferences.