Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values ( K 0 ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E / N ; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method‐dependent results) only if the gas nature, temperature or E / N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

In the present paper, theoretical simulations and experimental observations are used to describe the ion dynamics in a trapped ion mobility spectrometer. In particular, the ion motion, ion transmission and mobility separation are discussed as a function of the bath gas velocity, radial confinement, analysis time and speed. Mobility analysis and calibration procedure are reported for the case of sphere-like molecules for positive and negative ion modes. Results showed that a maximal mobility resolution can be achieved by optimizing the gas velocity, radial confinement (RF amplitude) and ramp speed (voltage range and ramp time). The mobility resolution scales with the electric field and gas velocity and R = 100–250 can be routinely obtained at room temperature.

Trapped 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.

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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.

There is currently a strong interest in the use of ion mobility spectrometry-mass spectrometry (IMS-MS) instrumentation for structural biology. In these applications, momentum transfer cross sections derived from IMS-MS measurements are used to reconstruct the three-dimensional analyte structure. Recent reports indicate that additional structural information can be extracted from measuring changes in cross sections in response to changes of the analyte structure. To further this approach, we constructed a tandem trapped IMS analyser (TIMS-TIMS) and incorporated it in a QqTOF mass spectrometer. TIMS-TIMS is constructed by coupling two TIMS analysers via an "interface region" composed of two apertures. We show that peptide oligomers (bradykinin) and native-like protein (ubiquitin) ions can be preserved through the course of an experiment in a TIMS-TIMS analyser. We demonstrate the ability to collisionally-activate as well as to trap mobility-selected ions, followed by subsequent mobility-analysis. In addition to inducing conformational changes, we show that we can fragment low charge states of ubiquitin at >1 mbar between the TIMS analysers with significant sequence coverage. Many fragment ions exhibit multiple features in their TIMS spectra, which means that they may not generally exist as the most stable isomer. The ability of TIMS-TIMS to dissociate mobility-selected protein ions and to measure the cross sections of their fragment ions opens new possibilities for IMS-based structure elucidation.

Glycoproteins play a central role in many biological processes including disease mechanisms. Nevertheless, because glycoproteins are heterogeneous entities, it remains unclear how glycosylation modulates the protein structure and function. Here, we assess the ability of tandem-trapped ion mobility spectrometry–mass spectrometry (tandem-TIMS/MS) to characterize the structure and sequence of the homotetrameric glycoprotein avidin. We show that (1) tandem-TIMS/MS retains native-like avidin tetramers with deeply buried solvent particles; (2) applying high activation voltages in the interface of tandem-TIMS results in collision-induced dissociation (CID) of avidin tetramers into compact monomers, dimers, and trimers with cross sections consistent with X-ray structures and reports from surface-induced dissociation (SID); (3) avidin oligomers are best described as heterogeneous ensembles with (essentially) random combinations of monomer glycoforms; (4) native top-down sequence analysis of the avidin tetramer is possible by CID in tandem-TIMS. Overall, our results demonstrate that tandem-TIMS/MS has the potential to correlate individual proteoforms to variations in protein structure.

The present work employs trapped ion mobility spectrometry (TIMS) for the analysis of ubiquitin ions known to display a multitude of previously unresolved interchangeable conformations upon electrospray ionization. The conformational distributions of ubiquitin [M + 6H](6+) through [M + 13H](13+) ions observed by TIMS are nearly identical to numerous drift tube ion mobility spectrometry studies reported in the literature. At an experimental resolving power up to ∼300, many of the congested conformations within the well-known compact, partially folded, and elongated [M + 7H](7+) states are separated. Minimizing the voltages (RF and DC) in the entrance funnel results in exclusive observation of compact [M + 7H](7+) conformers. However, under these conditions, the mobility-dependent pseudopotential coefficient may discriminate against ions having larger collision cross sections-a universal effect for all RF ion guides, funnels, and traps operating in the presence of a gas. The data presented underscore the complications associated with direct comparison of collision cross section values that represent an ensemble average of multiple underlying conformations. As illustrated herein, the microheterogeneity within a particular conformational family and the relative state-to-state abundance can be altered by solvent memory, energetic, and kinetic effects.