Recommendations for reporting ion mobility mass spectrometry measurements

© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. 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 (K0) 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

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 (<i>R</i>) 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 <i>R</i> 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

Isomerization Kinetics of AT Hook Decapeptide Solution Structures

The mammalian high mobility group protein HMGA2 contains three DNA binding motifs associated with many physiological functions including oncogenesis, obesity, stem cell youth, human height, and human intelligence. In the present paper, trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) has been utilized to study the conformational dynamics of the third DNA binding motif using the “AT hook” decapeptide unit (Lys¹-Arg²-Prol³-Arg⁴-Gly⁵-Arg⁶-Prol⁷-Arg⁸-Lys⁹-Trp¹⁰, ATHP) as a function of the solvent state. Solvent state distributions were preserved during electrospray ion formation, and multiple IMS bands were identified for the [M + 2H]²⁺ and for the [M + 3H]³⁺ charge states. Conformational isomer interconversion rates were measured as a function of the trapping time for the [M + 2H]²⁺ and [M + 3H]³⁺ charge states. Candidate structures were proposed for all IMS bands observed. Protonation site, proline residue conformation, and side chain orientations were identified as the main motifs governing the conformational interconversion processes. Conformational dynamics from the solvent state distribution to the gas-phase “de-solvated” state distribution demonstrated that ATHP is “structured”, and relative abundances are associated with the relative stability between the proposed conformers. The most stable ATHP [M + 2H]²⁺ conformation at the “de-solvated” state corresponds to the AT hook motif observed in AT-rich DNA regions

Isomerization Kinetics of AT Hook Decapeptide Solution Structures

The mammalian high mobility group protein HMGA2 contains three DNA binding motifs associated with many physiological functions including oncogenesis, obesity, stem cell youth, human height, and human intelligence. In the present paper, trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) has been utilized to study the conformational dynamics of the third DNA binding motif using the “AT hook” decapeptide unit (Lys¹-Arg²-Prol³-Arg⁴-Gly⁵-Arg⁶-Prol⁷-Arg⁸-Lys⁹-Trp¹⁰, ATHP) as a function of the solvent state. Solvent state distributions were preserved during electrospray ion formation, and multiple IMS bands were identified for the [M + 2H]²⁺ and for the [M + 3H]³⁺ charge states. Conformational isomer interconversion rates were measured as a function of the trapping time for the [M + 2H]²⁺ and [M + 3H]³⁺ charge states. Candidate structures were proposed for all IMS bands observed. Protonation site, proline residue conformation, and side chain orientations were identified as the main motifs governing the conformational interconversion processes. Conformational dynamics from the solvent state distribution to the gas-phase “de-solvated” state distribution demonstrated that ATHP is “structured”, and relative abundances are associated with the relative stability between the proposed conformers. The most stable ATHP [M + 2H]²⁺ conformation at the “de-solvated” state corresponds to the AT hook motif observed in AT-rich DNA regions

Separation and Identification of Isomeric Glycans by Selected Accumulation-Trapped Ion Mobility Spectrometry-Electron Activated Dissociation Tandem Mass Spectrometry

One of the major challenges in structural characterization of oligosaccharides is the presence of many structural isomers in most naturally occurring glycan mixtures. Although ion mobility spectrometry (IMS) has shown great promise in glycan isomer separation, conventional IMS separation occurs on the millisecond time scale, largely restricting its implementation to fast time-of-flight (TOF) analyzers which often lack the capability to perform electron activated dissociation (ExD) tandem MS analysis and the resolving power needed to resolve isobaric fragments. The recent development of trapped ion mobility spectrometry (TIMS) provides a promising new tool that offers high mobility resolution and compatibility with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometers when operated under the selected accumulation-TIMS (SA-TIMS) mode. Here, we present our initial results on the application of SA-TIMS-ExD-FTICR MS to the separation and identification of glycan linkage isomers

Direct Observation of Differences of Carotenoid Polyene Chain <i>cis</i>/<i>trans</i> Isomers Resulting from Structural Topology

In the present paper, trapped ion mobility spectrometry (TIMS) and theoretical calculations have been used to study carotenoid geometrical motifs generated by photoisomerization from the <i>all-trans</i> geometry. Multiple geometric isomers of the carotenoids lutein and zeaxanthin were separated using TIMS (R > 110) for [M]⁺, [M + H]⁺, and [M – 18]⁺ molecular species. Comparison of observed cross sections with those obtained from molecular dynamics calculations showed that the number of <i>cis</i> double bonds and <i>s-cis single bonds</i> in the polyene chain determine the topology space of the carotenoid. The intensities of IMS signals are correlated with the relative stability of these geometric isomers., The most stable isomer is the <i>all-trans</i> geometry regardless of the ionization state ([M – 18]⁺, [M]⁺, and [M + H]⁺), and structural stability decreases with the increasing number of <i>cis</i> and/or <i>s-cis</i> bonds in the polyene chain

Analysis of Photoirradiated Water Accommodated Fractions of Crude Oils Using Tandem TIMS and FT-ICR MS

For the first time, trapped ion mobility spectrometry (TIMS) in tandem with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is applied to the analysis of the low energy water accommodated fraction (WAF) of a crude oil as a function of the exposure to light. The TIMS-FT-ICR MS analysis provided, in addition to the heteroatom series identification, new insights into the WAF isomeric complexity (e.g., [<i>m</i>/<i>z</i>; chemical formula; collision cross section] data sets) for a better evaluation of the degree of chemical and structural photoinduced transformations. Inspection of the [<i>m</i>/<i>z</i>; chemical formula; collision cross section] data sets shows that the WAF composition changes as a function of the exposure to light in the first 115 h by initial photosolubilization of HC components and their photo-oxidation up to O_4–5 of mainly high double bond equivalence species (DBE > 9). The addition of high resolution TIMS (resolving power of 90–220) to ultrahigh resolution FT-ICR MS (resolving power over 400k) permitted the identification of a larger number of molecular components in a single analysis (e.g., over 47k using TIMS-MS compared to 12k by MS alone), with instances of over 6-fold increase in the number of molecular features per nominal mass due to the WAF isomeric complexity. This work represents a stepping stone toward a better understanding of the WAF components and highlights the need for better experimental and theoretical approaches to characterize the WAF structural diversity

Characterization of Intramolecular Interactions of Cytochrome <i>c</i> Using Hydrogen–Deuterium Exchange-Trapped Ion Mobility Spectrometry–Mass Spectrometry and Molecular Dynamics

Globular proteins, such as cytochrome <i>c</i> (cyt <i>c</i>), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt <i>c</i> was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time-resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry–mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the −5 to −10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt <i>c</i> major structural unfolding is associated with the distancing of the N- and C-terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity

Flavin Adenine Dinucleotide Structural Motifs: From Solution to Gas Phase

Flavin adenine dinucleotide (FAD) is involved in important metabolic reactions where the biological function is intrinsically related to changes in conformation. In the present work, FAD conformational changes were studied in solution and in gas phase by measuring the fluorescence decay time and ion-neutral collision cross sections (CCS, in a trapped ion mobility spectrometer, TIMS) as a function of the solvent conditions (i.e., organic content) and gas-phase collisional partner (i.e., N₂ doped with organic molecules). Changes in the fluorescence decay suggest that FAD can exist in four conformations in solution, where the abundance of the extended conformations increases with the organic content. TIMS-MS experiments showed that FAD can exist in the gas phase as deprotonated (M = C₂₇H₃₁N₉O₁₅P₂) and protonated forms (M = C₂₇H₃₃N₉O₁₅P₂) and that multiple conformations (up to 12) can be observed as a function of the starting solution for the [M + H]⁺ and [M + Na]⁺molecular ions. In addition, changes in the relative abundances of the gas-phase structures were observed from a “stack” to a “close” conformation when organic molecules were introduced in the TIMS cell as collision partners. Candidate structures optimized at the DFT/B3LYP/6-31G­(d,p) were proposed for each IMS band, and results showed that the most abundant IMS band corresponds to the most stable candidate structure. Solution and gas-phase experiments suggest that the driving force that stabilizes the different conformations is based on the interaction of the adenine and isoalloxazine rings that can be tailored by the “solvation” effect created with the organic molecules

Characterization of Intramolecular Interactions of Cytochrome <i>c</i> Using Hydrogen–Deuterium Exchange-Trapped Ion Mobility Spectrometry–Mass Spectrometry and Molecular Dynamics

Globular proteins, such as cytochrome <i>c</i> (cyt <i>c</i>), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt <i>c</i> was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time-resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry–mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the −5 to −10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt <i>c</i> major structural unfolding is associated with the distancing of the N- and C-terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity