Publications by authors named "Kevin Giles"

37 Publications

LESA Cyclic Ion Mobility Mass Spectrometry of Intact Proteins from Thin Tissue Sections.

Anal Chem 2020 05 27;92(9):6321-6326. Epub 2020 Apr 27.

Department of Chemistry and Biochemistry, Florida International University, Miami, Florida 33199, United States.

Liquid extraction surface analysis (LESA) is an ambient surface sampling technique that allows the analysis of intact proteins directly from tissue samples via mass spectrometry. Integration of ion mobility separation to LESA mass spectrometry workflows has shown significant improvements in the signal-to-noise ratios of the resulting protein mass spectra and hence the number of proteins detected. Here, we report the use of a quadrupole-cyclic ion mobility-time-of-flight mass spectrometer (Q-cIM-ToF) for the analysis of proteins from mouse brain and rat kidney tissues sampled via LESA. Among other features, the instrument allows multiple pass cyclic ion mobility separation, with concomitant increase in resolving power. Single-pass experiments enabled the detection of 30 proteins from mouse brain tissue, rising to 44 when quadrupole isolation was employed. In the absence of ion mobility separation, 21 proteins were detected in rat kidney tissue including the abundant α- and β-globin chains from hemoglobin. Single-pass cyclic ion mobility mass spectrometry enabled the detection of 60 additional proteins. Multipass experiments of a narrow / range (/ 870-920) resulted in the detection of 24 proteins (one pass), 37 proteins (two passes) and 54 proteins (three passes), thus demonstrating the benefits of improved mobility resolving power.
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http://dx.doi.org/10.1021/acs.analchem.9b05169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304663PMC
May 2020

A Novel Ion Pseudo-trapping Phenomenon within Traveling Wave Ion Guides.

J Am Soc Mass Spectrom 2020 Apr 5;31(4):880-887. Epub 2020 Mar 5.

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States.

The widespread use of traveling wave ion mobility (TWIM) technology in fields such as omics and structural biology motivates efforts to deepen our understanding of ion transport within such devices. Here, we describe a new advancement in TWIM theory, where pseudo-trapping within TW ion guides is characterized in detail. During pseudo-trapping, ions with different mobilities can travel with the same mean velocity, leaving others within the same TWIM experiment to separate as normal. Furthermore, pseudo-trapping limits typical band broadening experienced by ions during TWIM, manifesting as peaks with apparently improved IM resolving power, but all ions that undergo pseudo trapping are unable to separate by IM. SIMION simulations show that ions become locked into a repeated pattern of motion with respect to the TW reference frame during pseudo-trapping. We developed a simplified model capable of reproducing TW pseudo-trapping and reproducing trends observed in experimental data. Our model and simulations suggest that pseudo-trapping occurs only during experiments performed under static TWIM conditions, to an extent that depends on the detailed shape of the traveling wave. We show that pseudo-trapping alters the ion transit times and can adversely affect calibrated CCS measurements. Finally, we provide recommendations for avoiding unintentional pseudo-trapping in TWIM in order to obtain optimal separations and CCS determinations.
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http://dx.doi.org/10.1021/jasms.9b00095DOI Listing
April 2020

Collision Cross Sections of Charge-Reduced Proteins and Protein Complexes: A Database for Collision Cross Section Calibration.

Anal Chem 2020 03 27;92(6):4475-4483. Epub 2020 Feb 27.

Waters Corporation, Wilmslow, SK9 4AX, United Kingdom.

The use of charge-reducing reagents to generate lower-charge ions has gained popularity in the field of native mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS). This is because the lower number of charged sites decreases the propensity for Coulombic repulsions and unfolding/restructuring, helping to preserve the native-like structure. Furthermore, lowering the charge state consequently increases the mass-to-charge values (/), effectively increasing spacing between signals originating from small mass differences, such as different proteoforms or protein-drug complexes. IM-MS yields collision cross section (CCS, Ω) values that provide information about the three-dimensional structure of the ion. Traveling wave IM (TWIM) is an established and expanding technique within the native MS field. TWIM measurements require CCS calibration, which is achieved via the use of standard species of known CCS. Current databases for native-like proteins and protein complexes provide CCS values obtained using normal (i.e., non-charge-reducing) conditions. Herein, we explored the validity of using "normal" charge calibrants to calibrate for charge-reduced proteins and show cases where it is not appropriate. Using a custom linear field drift cell that enables the determination of ion mobilities from "first principles", we directly determined CCS values for 19 protein calibrant species under three solution conditions (yielding a broad range of charge states) and two drift gases. This has established a database of CCS and reduced-mobility () values, along with their associated uncertainties, for proteins and protein complexes over a large / range. TWIM validation of this database shows improved accuracy over existing methods in calibrating CCS values for charge-reduced proteins.
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http://dx.doi.org/10.1021/acs.analchem.9b05519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170229PMC
March 2020

Isolation of Crude Oil Peaks Differing by / ∼0.1 via Tandem Mass Spectrometry Using a Cyclic Ion Mobility-Mass Spectrometer.

Anal Chem 2019 11 29;91(22):14268-14274. Epub 2019 Oct 29.

Department of Chemistry , Kyungpook National University , 80 Daehak-ro, Buk-gu , Daegu 41566 , Republic of Korea.

Mass spectrometry is widely used in studying the structures of compounds present in crude oil. In this study, a novel mass spectrometer incorporating a cyclic ion mobility separator was used to obtain tandem mass spectra of crude oil compounds in a narrow mass-to-charge ratio ( window. Isolation of specific peaks was performed by combining quadrupole and ion mobility separation. As a result, peaks differing by an / value of 0.1 could be isolated. Tandem mass spectrometry with collision-induced dissociation was successfully performed to study the chemical structures of the isolated ions. A series of ions ranging from / 374 to / 384, differing by two hydrogen atoms but with the same number of carbons, were isolated and tandem mass spectra were obtained. The higher / precursor ions produced smaller fragment ions; this is explained by the reduced aromaticity owing to an increased number of hydrogen atoms. The ions at / 388 and 374, differing by a CH group, produced very similar fragmentation patterns. Overall, the data obtained from this study clearly demonstrate that the novel cyclic ion mobility-mass spectrometer is a powerful instrument that can provide tandem mass spectra of individual compounds constituting complex mixtures such as crude oils.
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http://dx.doi.org/10.1021/acs.analchem.9b02255DOI Listing
November 2019

Structure Determination of Large Isomeric Oligosaccharides of Natural Origin through Multipass and Multistage Cyclic Traveling-Wave Ion Mobility Mass Spectrometry.

Anal Chem 2019 09 3;91(18):12030-12037. Epub 2019 Sep 3.

INRA , UR1268 Biopolymers Interactions Assemblies, La Géraudière B.P. 71627 , F-44316 Nantes , France.

Carbohydrate isomers with identical atomic composition cannot be distinguished by mass spectrometry. By separating the ions according to their conformation in the gas phase, ion mobility (IM) coupled to mass spectrometry is an attractive approach to overcome this issue and extend the limits of mass spectrometry in structural glycosciences. Recent technological developments have significantly increased the resolving power of ion mobility separators. One such instrument features a cyclic traveling-wave IM separator integrated in a quadrupole/time-of-flight mass spectrometer. This system allows for multipass ion separations and for pre-, intra-, and post-IM fragmentation. In the present study, we utilize this system to explore a complex mixture of oligoporphyrans derived from the enzymatic digestion of the cell wall of the red alga . We are able to deduce their complete structure using IM arrival times and the / of specific fragments. This approach was successfully applied for sequencing of oligoporphyrans of up to 1500 Da and included the positioning of the methyl ether and sulfate groups. The structures defined in this study by IM-MS/MS agree with those found in the past but use much more time-consuming analytical approaches. This study also revealed some so far undescribed structures, present at very low abundance. In addition, the results made it possible to compare the abundance of the different isomers released by the enzyme and to draw further conclusions on the specificity of β-porphyranase and more particularly on its accommodation tolerance of anhydro-bridges in subsites. Finally, a separation of two isomers with very similar mobility was obtained after 58 passes around the cIM, with an estimated resolving power of 920 for these triply charged species, confirming the structures attributed to these two isomers.
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http://dx.doi.org/10.1021/acs.analchem.9b03036DOI Listing
September 2019

A Cyclic Ion Mobility-Mass Spectrometry System.

Anal Chem 2019 07 12;91(13):8564-8573. Epub 2019 Jun 12.

Waters Corporation , Stamford Avenue, Altrincham Road , Wilmslow SK9 4AX , U.K.

Improvements in the performance and availability of commercial instrumentation have made ion mobility-mass spectrometry (IM-MS) an increasingly popular approach for the structural analysis of ionic species as well as for separation of complex mixtures. Here, a new research instrument is presented which enables complex experiments, extending the current scope of IM technology. The instrument is based on a Waters SYNAPT G2-S i IM-MS platform, with the IM separation region modified to accept a cyclic ion mobility (cIM) device. The cIM region consists of a 98 cm path length, closed-loop traveling wave (TW)-enabled IM separator positioned orthogonally to the main ion optical axis. A key part of this geometry and its flexibility is the interface between the ion optical axis and the cIM, where a planar array of electrodes provides control over the TW direction and subsequent ion motion. On either side of the array, there are ion guides used for injection, ejection, storage, and activation of ions. In addition to single and multipass separations around the cIM, providing selectable mobility resolution, the instrument design and control software enable a range of "multifunction" experiments such as mobility selection, activation, storage, IMS , and importantly custom combinations of these functions. Here, the design and performance of the cIM-MS instrument is highlighted, with a mobility resolving power of approximately 750 demonstrated for 100 passes around the cIM device using a reverse sequence peptide pair. The multifunction capabilities are demonstrated through analysis of three isomeric pentasaccharide species and the small protein ubiquitin.
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http://dx.doi.org/10.1021/acs.analchem.9b01838DOI Listing
July 2019

Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Traveling Wave Device.

Anal Chem 2019 06 5;91(12):7554-7561. Epub 2019 Jun 5.

Institute of Structural and Molecular Biology, Division of Biosciences , University College London , London , WC1E 6BT , United Kingdom.

Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into "conformational families". Increasing the IM resolving power should allow finer structural information to be obtained and can be achieved by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas phase and previous experiments have shown that the initial conformations of small proteins can be lost within tens of milliseconds. Here, we report on investigations of protein ion stability using a multipass traveling wave (TW) cyclic IM (cIM) device. Using this device, minimal structural changes were observed for Cytochrome C after hundreds of milliseconds, while no changes were observed for a larger multimeric complex (Concanavalin A). The geometry of the instrument (Q-cIM-ToF) also enables complex tandem IM experiments to be performed, which were used to obtain more detailed collision-induced unfolding pathways for Cytochrome C. The instrument geometry provides unique capabilities with the potential to expand the field of protein analysis via IM-MS.
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http://dx.doi.org/10.1021/acs.analchem.8b05641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006968PMC
June 2019

Cyclic Ion Mobility Mass Spectrometry Distinguishes Anomers and Open-Ring Forms of Pentasaccharides.

J Am Soc Mass Spectrom 2019 Jun 11;30(6):1028-1037. Epub 2019 Apr 11.

Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, UK.

There is increasing biopharmaceutical interest in oligosaccharides and glycosylation. A key requirement for these sample types is the ability to characterize the chain length, branching, type of monomers, and importantly stereochemistry and anomeric configuration. Herein, we showcase the multi-function capability of a cyclic ion mobility (cIM) separator embedded in a quadrupole/time-of-flight mass spectrometer (Q-ToF MS). The instrument design enables selective activation of mobility-separated precursors followed by cIM separation of product ions, an approach analogous to MS. Using high cIM resolution, we demonstrate the separation of three isomeric pentasaccharides and, moreover, that three components are present for each compound. We show that structural differences between product ions reflect the precursor differences in some cases but not others. These findings are corroborated by a heavy oxygen labelling approach. Using this methodology, the identity of fragment ions may be assigned. This enables us to postulate that the two main components observed for each pentasaccharide are anomeric forms. The remaining low abundance component is assigned as an open-ring form.
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http://dx.doi.org/10.1007/s13361-019-02168-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517361PMC
June 2019

Recommendations for reporting ion mobility Mass Spectrometry measurements.

Mass Spectrom Rev 2019 05 1;38(3):291-320. Epub 2019 Feb 1.

CNRS, INSERM and University of Bordeaux, Institut Européen de Chimie et Biologie, Pessac, France.

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 ) 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.
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http://dx.doi.org/10.1002/mas.21585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618043PMC
May 2019

Investigations into the performance of travelling wave enabled conventional and cyclic ion mobility systems to characterise protomers of fluoroquinolone antibiotic residues.

Rapid Commun Mass Spectrom 2019 Jul 18;33 Suppl 2:11-21. Epub 2019 Feb 18.

Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, UK.

Rationale: Fluoroquinolones (FLQs) have been shown to form protomers with distinctive fragment profiles. Experimental parameters affect protomer formation, impacting observed conventional tandem mass spectrometric (MS/MS) dissociation and multiple reaction monitoring (MRM) transition reproducibility. Collision cross section (CCS) measurement can provide an additional identification metric and improved ion mobility (IM) separation strategies could provide further understanding of fluctuations in fragmentation when using electrospray ionisation (ESI).

Methods: Porcine muscle tissue was fortified with nine fluoroquinolone antibiotics. Extracts were cleaned using QuEChERS dispersive extraction. Separation was achieved via ultra-high-performance liquid chromatography (UHPLC) and analysis performed using positive ion ESI coupled with linear T-wave IM (N and CO drift gas) and cyclic IM-MS (calibrated to perform accurate mass and CCS measurement).

Results: IM-resolved protomeric species have been observed for nine FLQs (uniquely three for danofloxacin). Long-term reproducibility and cross-platform T-wave/cIM studies have demonstrated CCS metric errors <1.5% when compared with a FLQ protomer reference CCS library. When comparing FLQ protomer separation using a standard, linear T-wave IM separator (N /CO ) and using a high-resolution cyclic T-wave device (N ), protomer peak-to-peak resolution ranged between Rs = 1 to Rs = 6 for the IM strategies utilised.

Conclusions: CCS is a reliable cross platform metric; specific FLQ CCS identification fingerprints have been produced, illustrating the potential to compliment MS/MS specificity or provide an alternative identification metric. Using cIM there is opportunity to correlate the erratic nature of protomer formation with the analytical conditions used and to gain further understanding of ionisation/dissociation mechanisms taking place during routine analyses.
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http://dx.doi.org/10.1002/rcm.8371DOI Listing
July 2019

A Mass-Spectrometry-Based Modelling Workflow for Accurate Prediction of IgG Antibody Conformations in the Gas Phase.

Angew Chem Int Ed Engl 2018 12 27;57(52):17194-17199. Epub 2018 Nov 27.

Department of Chemistry, King's College London, 7 Trinity Street, London, SE1 1DB, UK.

Immunoglobulins are biomolecules involved in defence against foreign substances. Flexibility is key to their functional properties in relation to antigen binding and receptor interactions. We have developed an integrative strategy combining ion mobility mass spectrometry (IM-MS) with molecular modelling to study the conformational dynamics of human IgG antibodies. Predictive models of all four human IgG subclasses were assembled and their dynamics sampled in the transition from extended to collapsed state during IM-MS. Our data imply that this collapse of IgG antibodies is related to their intrinsic structural features, including Fab arm flexibility, collapse towards the Fc region, and the length of their hinge regions. The workflow presented here provides an accurate structural representation in good agreement with the observed collision cross section for these flexible IgG molecules. These results have implications for studying other nonglobular flexible proteins.
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http://dx.doi.org/10.1002/anie.201812018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392142PMC
December 2018

Identifying key membrane protein lipid interactions using mass spectrometry.

Nat Protoc 2018 05 26;13(5):1106-1120. Epub 2018 Apr 26.

Department of Chemistry, University of Oxford, Oxford, UK.

With the recent success in determining membrane protein structures, further detailed understanding of the identity and function of the bound lipidome is essential. Using an approach that combines high-energy native mass spectrometry (HE-nMS) and solution-phase lipid profiling, this protocol can be used to determine the identity of the endogenous lipids that directly interact with a protein. Furthermore, this method can identify systems in which such lipid binding has a major role in regulating the oligomeric assembly of membrane proteins. The protocol begins with recording of the native mass spectrum of the protein of interest, under successive delipidation conditions, to determine whether delipidation leads to disruption of the oligomeric state. Subsequently, we propose using a bipronged strategy: first, an HE-nMS platform is used that allows dissociation of the detergent micelle at the front end of the instrument. This allows for isolation of the protein-lipid complex at the quadrupole and successive fragmentation at the collision cell, which leads to identification of the bound lipid masses. Next, simultaneous coupling of this with in-solution LC-MS/MS-based identification of extracted lipids reveals the complete identity of the interacting lipidome that copurifies with the proteins. Assimilation of the results of these two sets of experiments divulges the complete identity of the set of lipids that directly interact with the membrane protein of interest, and can further delineate its role in maintaining the oligomeric state of the protein. The entire procedure takes 2 d to complete.
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http://dx.doi.org/10.1038/nprot.2018.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049616PMC
May 2018

Multiple Gas-Phase Conformations of a Synthetic Linear Poly(acrylamide) Polymer Observed Using Ion Mobility-Mass Spectrometry.

J Am Soc Mass Spectrom 2017 11 14;28(11):2492-2499. Epub 2017 Aug 14.

Mass Spectrometry Laboratory, University of Liège, Quartier Agora, Allée du Six Aout 11, B-4000, Liège, Belgium.

Ion mobility-mass spectrometry (IM-MS) has emerged as a powerful separation and identification tool to characterize synthetic polymer mixtures and topologies (linear, cyclic, star-shaped,…). Electrospray coupled to IM-MS already revealed the coexistence of several charge state-dependent conformations for a single charge state of biomolecules with strong intramolecular interactions, even when limited resolving power IM-MS instruments were used. For synthetic polymers, the sample's polydispersity allows the observation of several chain lengths. A unique collision cross-section (CCS) trend is usually observed when increasing the degree of polymerization (DP) at constant charge state, allowing the deciphering of different polymer topologies. In this paper, we report multiple coexisting CCS trends when increasing the DP at constant charge state for linear poly(acrylamide) PAAm in the gas phase. This is similar to observations on peptides and proteins. Biomolecules show in addition population changes when collisionally heating the ions. In the case of synthetic PAAm, fragmentation occurred before reaching the energy for conformation conversion. These observations, which were made on two different IM-MS instruments (SYNAPT G2 HDMS and high resolution multi-pass cyclic T-Wave prototype from Waters), limit the use of ion mobility for synthetic polymer topology interpretations to polymers where unique CCS values are observed for each DP at constant charge state. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1769-xDOI Listing
November 2017

Ozone-induced dissociation on a traveling wave high-resolution mass spectrometer for determination of double-bond position in lipids.

Rapid Commun Mass Spectrom 2017 Sep;31(17):1415-1423

Department of Chemistry & Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27412, USA.

Rationale: The position of C=C within fatty acyl chains affects the biological function of lipids. Ozone-induced dissociation mass spectrometry (OzID-MS) has great potential in determination of lipid double-bond position, but has generally been implemented on low-resolution ion trap mass spectrometers. In addition, most of the OzID-MS experiments carried out so far were focused on the sodiated adducts of lipids; fragmentation of the most commonly observed protonated ions generated in LC/MS-based lipidomics workflow has been less explored.

Methods: Ozone generated in line from an ozone generator was connected to the trap and transfer gas supply line of a Synapt G2 high-resolution mass spectrometer. Protonated ions of different phosphatidylcholines (PC) were generated by electrospray ionization through direct infusion. Different parameters, including traveling wave height and velocity, trap entrance and DC potential, were adjusted to maximize the OzID efficiency. sn-positional isomers and cis/trans isomers of lipids were compared for their reactivity with ozone.

Results: Traveling wave height and velocity were tuned to prolong the encounter time between lipid ions and ozone, and resulted in improved OzID efficiency, as did increasing trapping region DC and entrance potential. Under optimized settings, at least 1000 times enhancement in OzID efficiency was achieved compared to that under default settings for monounsaturated PC standards. Monounsaturated C=C in the sn-2 PC isomer reacted faster with ozone than the sn-1 isomer. Similarly, the C=C in trans PC reacted faster than in cis PC.

Conclusions: This is the first implementation of OzID in the trap and transfer region of a traveling wave enabled high-resolution mass spectrometer. The OzID reaction efficiency is significantly improved by slowing down ions in the trap region for their prolonged interaction with ozone. This will facilitate application of high-resolution OzID-MS in lipidomics.
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http://dx.doi.org/10.1002/rcm.7920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560992PMC
September 2017

New High Resolution Ion Mobility Mass Spectrometer Capable of Measurements of Collision Cross Sections from 150 to 520 K.

Anal Chem 2016 10 15;88(19):9469-9478. Epub 2016 Sep 15.

Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute for Biotechnology, University of Manchester , Manchester M1 7DN, U.K.

We present a new variable temperature (VT), high resolution ion mobility (IM) drift tube coupled to a commercial mass spectrometer (MS). Ions are generated in an electrospray ion source with a sampling cone interface and two stacked ring RF guides which transfer ions into the mobility analyzer located prior to a quadrupole time-of-flight mass spectrometer. The drift cell can be operated over a pressure range of 0.5-3 Torr and a temperature range of 150-520 K with applied fields typically between 3 and 14 V cm. This makes the instrument suitable for rotationally averaged collision cross section (CCS) measurements at low E/N ratios where ions are near thermal equilibrium with the buffer gas. Fundamental studies of the effective ion temperatures can be performed at high E/N ratios. An RF ion trap/buncher is located at the beginning of the drift region, which modulates the continuous ion beam into spatially narrow packets. Packets of ions then drift in a linear electric field, which is 50.5 cm long, and are separated according to their mobility in an inert buffer gas. Post-drift, an ion funnel focuses the radially spread pulses of ions into the inlet of a commercial MS platform (Micromass QToF2). We present the novel features of this instrument and results from VT-IM-MS experiments on a range of model systems-IMS CCS standards (Agilent ESI Tune Mix), the monomeric protein Ubiquitin (8.6 kDa), and the tetrameric protein complex Concanavalin A (103 kDa). We evaluate the performance of the instrument by comparing ambient CCS values of model compounds with those found in the literature. Several effects of temperature on collision cross sections and resolution are observed. For small rigid molecules, changes in resolution are consistent with anticipated thermal diffusion effects. Changes in measured CCS for these rigid systems at different temperatures are attributed primarily to the effect of temperature on the long-range attractive interaction. Similar effects are seen for protein ions at low temperatures, although there is also some evidence for structural transitions. By heating the protein ions, their conformational profiles are significantly altered. Very high temperatures narrow the conformational space presented by both Ubiquitin and Concanavalin; it appears that diverse conformational families are "melted" into more homogeneous populations. Because of this conformational heterogeneity, the apparent IMS resolution obtained for proteins at ambient and reduced temperatures is an order of magnitude lower than the expected diffusion limited resolution (R). This supports a hypothesis that the broad CCS features frequently observed for proteins do not correspond to interconverting conformers, but rather to high numbers of intrinsically stable structures.
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http://dx.doi.org/10.1021/acs.analchem.6b01812DOI Listing
October 2016

Combining density functional theory (DFT) and collision cross-section (CCS) calculations to analyze the gas-phase behaviour of small molecules and their protonation site isomers.

Analyst 2016 Jun;141(13):4044-54

Biomolecular & Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, Antwerp, Belgium. and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK and School of Molecular and Cellular Biology, University of Leeds, LS2 9JT, UK.

Electrospray ion mobility-mass spectrometry (IM-MS) data show that for some small molecules, two (or even more) ions with identical sum formula and mass, but distinct drift times are observed. In spite of showing their own unique and characteristic fragmentation spectra in MS/MS, no configurational or constitutional isomers are found to be present in solution. Instead the observation and separation of such ions appears to be inherent to their gas-phase behaviour during ion mobility experiments. The origin of multiple drift times is thought to be the result of protonation site isomers ('protomers'). Although some important properties of protomers have been highlighted by other studies, correlating the experimental collision cross-sections (CCSs) with calculated values has proven to be a major difficulty. As a model, this study uses the pharmaceutical compound melphalan and a number of related molecules with alternative (gas-phase) protonation sites. Our study combines density functional theory (DFT) calculations with modified MobCal methods (e.g. nitrogen-based Trajectory Method algorithm) for the calculation of theoretical CCS values. Calculated structures can be linked to experimentally observed signals, and a strong correlation is found between the difference of the calculated dipole moments of the protomer pairs and their experimental CCS separation.
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http://dx.doi.org/10.1039/c5an02456kDOI Listing
June 2016

Ion mobility mass spectrometry of peptide, protein, and protein complex ions using a radio-frequency confining drift cell.

Analyst 2016 Feb 7;141(3):884-91. Epub 2016 Jan 7.

University of Washington, Department of Chemistry, Box 351700, Seattle, WA 98195-1700, USA.

Ion mobility mass spectrometry experiments enable the characterization of mass, assembly, and shape of biological molecules and assemblies. Here, a new radio-frequency confining drift cell is characterized and used to measure the mobilities of peptide, protein, and protein complex ions. The new drift cell replaced the traveling-wave ion mobility cell in a Waters Synapt G2 HDMS. Methods for operating the drift cell and determining collision cross section values using this experimental set up are presented within the context of the original instrument control software. Collision cross sections for 349 cations and anions are reported, 155 of which are for ions that have not been characterized previously using ion mobility. The values for the remaining ions are similar to those determined using a previous radio-frequency confining drift cell and drift tubes without radial confinement. Using this device under 2 Torr of helium gas and an optimized drift voltage, denatured and native-like ions exhibited average apparent resolving powers of 14.2 and 16.5, respectively. For ions with high mobility, which are also low in mass, the apparent resolving power is limited by contributions from ion gating. In contrast, the arrival-time distributions of low-mobility, native-like ions are not well explained using only contributions from ion gating and diffusion. For those species, the widths of arrival-time distributions are most consistent with the presence of multiple structures in the gas phase.
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http://dx.doi.org/10.1039/c5an02107cDOI Listing
February 2016

UV photodissociation of trapped ions following ion mobility separation in a Q-ToF mass spectrometer.

Analyst 2014 Dec;139(24):6348-51

Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute for Biotechnology, University of Manchester, UK.

An ion mobility mass spectrometer has been modified to allow optical interrogation of ions with different mass-to-charge (m/z) ratios and/or mobilities (K). An ion gating and trapping procedure has been developed which allows us to store ions for several seconds enabling UV photodissociation (UVPD).
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http://dx.doi.org/10.1039/c4an01656dDOI Listing
December 2014

Elucidating molecular structures of nonalkylated and short-chain alkyl (n < 5, (CH₂)n) aromatic compounds in crude oils by a combination of ion mobility and ultrahigh-resolution mass spectrometries and theoretical collisional cross-section calculations.

Anal Chem 2014 Apr 18;86(7):3300-7. Epub 2014 Mar 18.

Kyungpook National University , Department of Chemistry, Daegu, 702-701 Republic of Korea.

Ultrahigh-resolution mass spectrometry has allowed the determination of elemental formulas of the compounds comprising crude oils. However, elucidating molecular structures remains an analytical challenge. Herein, we propose and demonstrate an approach combining ion mobility mass spectrometry (IM-MS), ultrahigh-resolution mass spectrometry, and theoretical collisional cross-section (CCS) calculations to determine the molecular structures of aromatic compounds found in crude oils. The approach is composed of three steps. First, chemical structures are suggested based on the elemental formulas determined from ultrahigh-resolution mass spectra. Second, theoretical CCS values are calculated based on these proposed structures. Third, the calculated CCS values of the proposed structures are compared with experimentally determined CCS values from IM-MS data to provide proposed structures. For proof of concept, 31 nonalkylated and short-chain alkyl (n < 5, (CH2)n) aromatic compounds commonly observed in crude oils were analyzed. Theoretical and experimental CCS values matched within a 5% RMS error. This approach was then used to propose structures of compounds in selected m/z regions of crude oil samples. Overall, the combination of ion mobility mass spectrometry, ultrahigh-resolution mass spectrometry, and theoretical calculations was shown to be a useful tool for elucidating chemical structures of compounds in complex mixtures.
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http://dx.doi.org/10.1021/ac4032737DOI Listing
April 2014

Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase.

Nucleic Acids Res 2013 May 21;41(9):5115-26. Epub 2013 Mar 21.

School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ' Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ' complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg(2+) conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3.
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http://dx.doi.org/10.1093/nar/gkt173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3643586PMC
May 2013

QconCAT standard for calibration of ion mobility-mass spectrometry systems.

J Proteome Res 2012 Nov 9;11(11):5564-72. Epub 2012 Oct 9.

Manchester Institute of Biotechnology, Michael Barber Centre for Mass Spectrometry, School of Chemistry, University of Manchester, Manchester, M1 7DN, United Kingdom.

Ion mobility-mass spectrometry (IM-MS) is a useful technique for determining information about analyte ion conformation in addition to mass/charge ratio. The physical principles that govern the mobility of an ion through a gas in the presence of a uniform electric field are well understood, enabling rotationally averaged collision cross sections (Ω) to be directly calculated from measured drift times under well-defined experimental conditions. However, such "first principle" calculations are not straightforward for Traveling Wave (T-Wave) mobility separations due to the range of factors that influence ion motion through the mobility cell. If collision cross section information is required from T-Wave mobility separations, then calibration of the instruments using known standards is essential for each set of experimental conditions. To facilitate such calibration, we have designed and generated an artificial protein based on the QconCAT technology, QCAL-IM, which upon proteolysis can be used as a universal ion mobility calibration standard. This single unique standard enables empirical calculation of peptide ion collision cross sections from the drift time on a T-Wave mobility instrument.
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http://dx.doi.org/10.1021/pr3005327DOI Listing
November 2012

Resolving structural isomers of monosaccharide methyl glycosides using drift tube and traveling wave ion mobility mass spectrometry.

Anal Chem 2012 Apr 13;84(7):3231-9. Epub 2012 Mar 13.

Department of Chemistry, Washington State University, Pullman, Washington 99164, USA.

Monosaccharide structural isomers including sixteen methyl-D-glycopyranosides and four methyl-N-acetylhexosamines were subjected to ion mobility measurements by electrospray ion mobility mass spectrometry. Two ion mobility-MS systems were employed: atmospheric pressure drift tube ion mobility time-of-flight mass spectrometry and a Synapt G2 HDMS system which incorporates a low pressure traveling wave ion mobility separator. All the compounds were investigated as [M + Na](+) ions in the positive mode. A majority of the monosaccharide structural isomers exhibited different mobility drift times in either system, depending on differences in their anomeric and stereochemical configurations. In general, drift time patterns (relative drift times of isomers) matched between the two instruments. Higher resolving power was observed using the atmospheric pressure drift tube. Collision cross section values of monosaccharide structural isomers were directly calculated from the atmospheric pressure ion mobility experiments, and a collision cross section calibration curve was made for the traveling wave ion mobility instrument. Overall, it was demonstrated that ion mobility-mass spectrometry using either drift tube or traveling wave ion mobility is a valuable technique for resolving subtle variations in stereochemistry among the sodium adducts of monosaccharide methyl glycosides.
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http://dx.doi.org/10.1021/ac203116aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633476PMC
April 2012

Nanospray ion mobility mass spectrometry of selected high mass species.

Methods Mol Biol 2011 ;790:57-70

Waters Corporation, Manchester, UK.

The introduction of electrospray ionization (ESI) and in particular nano-electrospray (nESI) has enabled the routine mass spectrometric (MS) analysis of large protein complexes in native aqueous buffers. Time-of-flight (ToF) mass spectrometers, in particular the hybrid quadrupole time-of-flight (Q-ToF) instruments, are well suited to the analysis of large protein complexes. When ionized under native-MS conditions, protein complexes routinely exhibit multiple charge states in excess of m/z 6,000, well above the standard mass range of many quadrupole or ion cyclotron-based instruments. The research area of native MS has expanded considerably in the last decade and has shown particular relevance in the area of protein structure determination. Researchers are now able to routinely measure intact MS spectra of protein complexes above 1 MDa in mass. The advent of ion mobility mass spectrometry (IM-MS), in combination with molecular dynamics (MD) studies, is now allowing researchers to infer the shape of the protein complex being analyzed. Herein, we describe how to acquire IM-MS data that ranges from inorganic salt clusters of caesium iodide (CsI) to large biomolecular complexes such as the chaperone protein GroEL.
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http://dx.doi.org/10.1007/978-1-61779-319-6_5DOI Listing
January 2012

Enhancements in travelling wave ion mobility resolution.

Rapid Commun Mass Spectrom 2011 Jun;25(11):1559-66

Waters Corporation, MS Technologies Centre, Floats Road, Manchester M23 9LZ, UK.

The use of ion mobility separation to determine the collision cross-section of a gas-phase ion can provide valuable structural information. The introduction of travelling-wave ion mobility within a quadrupole/time-of-flight mass spectrometer has afforded routine collision cross-section measurements to be performed on a range of ionic species differing in gas-phase size/structure and molecular weight at physiologically relevant concentrations. Herein we discuss the technical advances in the second-generation travelling-wave ion mobility separator, which result in up to a four-fold increase in mobility resolution. This improvement is demonstrated using two reverse peptides (mw 490 Da), small ruthenium-containing anticancer drugs (mw 427 Da), a cisplatin-modified protein (mw 8776 Da) and the noncovalent tetradecameric chaperone complex GroEL (mw 802 kDa). What is also shown are that the collision cross-sections determined using the second-generation mobility separator correlate well with the previous generation and theoretically derived values.
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http://dx.doi.org/10.1002/rcm.5013DOI Listing
June 2011

Ion mobility separation coupled with MS detects two structural states of Alzheimer's disease Aβ1-40 peptide oligomers.

J Mol Biol 2011 Mar 13;407(1):110-24. Epub 2011 Jan 13.

Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

Mounting evidence points to the soluble oligomers of amyloid β (Aβ) peptide as important neurotoxic species in Alzheimer's disease, causing synaptic dysfunction and neuronal injury, and finally leading to neuronal death. The mechanism of the Aβ peptide self-assembly is still under debate. Here, Aβ1-40 peptide oligomers were studied using mass spectrometry combined with ion mobility spectrometry, which allowed separation of the signals of numerous oligomers and measurement of their collisional cross-section values (Ω). For several oligomers, at least two different species of different Ω values were detected, indicating the presence of at least two families of conformers: compact and extended. The obtained results are rationalized by a set of molecular models of Aβ1-40 oligomer structure that provided a very good correlation between the experimental and theoretical Ω values, both for the compact and the extended forms. Our results indicate that mass spectrometry detects oligomeric species that are on-pathway in the process of fibril formation or decay, but also alternative structures which may represent off-pathway evolution of oligomers.
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http://dx.doi.org/10.1016/j.jmb.2011.01.012DOI Listing
March 2011

Application of the Mason-Schamp equation and ion mobility mass spectrometry to identify structurally related compounds in crude oil.

Anal Chem 2011 Jan 30;83(1):77-83. Epub 2010 Nov 30.

Department of Chemistry, Kyungpook National University, Daegu, Korea.

The various components of crude oil were structurally resolved using an atmospheric-pressure solids analysis probe (ASAP) coupled with ion mobility mass spectrometry (IM-MS). An ASAP source was used to broadly fractionate compounds according to their boiling points, thereby simplifying the resulting mass spectra for easier data interpretation. The m/z-mobility plots obtained by IM-MS analysis of crude oil could be used to find the structural relationship between crude oil molecules. That was demonstrated using ion mobility mass spectra from a homologous series of compounds, differing only by the number of alkyl units, found in crude oil. The peaks from this series were linearly aligned in the plot, suggesting a continuous increase of the collisional cross section with an increase of mass values and hence the absence of significant structural differences within the series. In contrast, peaks in a homologous series differing only in the number of pendant hydrogen atoms were not linearly aligned, suggesting a discontinuous increase of the collisional cross section with an increase of mass values and hence significant structural differences due to the addition or removal of hydrogen. Cases in which a slope change was observed at three- or four-peak intervals may be related to the addition of an aromatic ring to existing structures. Overall, ion mobility mass spectrometry demonstrates a useful tool that can be used to elucidate structural relationships between molecules comprising crude oil.
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http://dx.doi.org/10.1021/ac101934qDOI Listing
January 2011

Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology.

Anal Chem 2010 Nov 27;82(22):9557-65. Epub 2010 Oct 27.

Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.

Collision cross sections in both helium and nitrogen gases were measured directly using a drift cell with RF ion confinement inserted within a quadrupole/ion mobility/time-of-flight hybrid mass spectrometer (Waters Synapt HDMS, Manchester, U.K.). Collision cross sections for a large set of denatured peptide, denatured protein, native-like protein, and native-like protein complex ions are reported here, forming a database of collision cross sections that spans over 2 orders of magnitude. The average effective density of the native-like ions is 0.6 g cm(-3), which is significantly lower than that for the solvent-excluded regions of proteins and suggests that these ions can retain significant memory of their solution-phase structures rather than collapse to globular structures. Because the measurements are acquired using an instrument that mimics the geometry of the commercial Synapt HDMS instrument, this database enables the determination of highly accurate collision cross sections from traveling-wave ion mobility data through the use of calibration standards with similar masses and mobilities. Errors in traveling-wave collision cross sections determined for native-like protein complexes calibrated using other native-like protein complexes are significantly less than those calibrated using denatured proteins. This database indicates that collision cross sections in both helium and nitrogen gases can be well-correlated for larger biomolecular ions, but non-correlated differences for smaller ions can be more significant. These results enable the generation of more accurate three-dimensional models of protein and other biomolecular complexes using gas-phase structural biology techniques.
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http://dx.doi.org/10.1021/ac1022953DOI Listing
November 2010

Isomer separation and gas-phase configurations of organoruthenium anticancer complexes: ion mobility mass spectrometry and modeling.

J Am Soc Mass Spectrom 2009 Jun 14;20(6):1119-22. Epub 2009 Feb 14.

Department of Chemistry, University of Warwick, Coventry, United Kingdom.

We have used ion mobility-mass spectrometry combined with molecular modeling for the separation and configurational analysis of three low-molecular-weight isomeric organoruthenium anticancer complexes containing ortho-, meta-, or para-terphenyl arene ligands. The isomers were separated using ion mobility based on traveling-wave technology and the experimentally determined collision cross sections were compared to theoretical calculations. Excellent agreement was observed between the experimentally and theoretically derived measurements.
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http://dx.doi.org/10.1016/j.jasms.2009.02.016DOI Listing
June 2009

Are liquid chromatography/electrospray tandem quadrupole fragmentation ratios unequivocal confirmation criteria?

Rapid Commun Mass Spectrom 2009 Apr;23(7):985-98

Official Food Control Authority of the Canton of Zurich (Kantonales Labor Zürich), P.O. Box, CH-8030 Zürich, Switzerland.

Multiple reaction monitoring (MRM) ratios as provided by tandem mass spectrometers are used to confirm positive residue findings (e.g. veterinary drugs or pesticides). The Commission Decision 2002/657/EEC defines tolerance levels for MRM ratios, which are intended to prevent the reporting of false positives. This paper reports findings where blank sample extracts have been spiked by a drug (difloxacin) and the corresponding measured MRM ratios significantly deviated from MRM ratios observed in matrix-free solution. The observation was explained by the formation of two different [M+H](+) analyte ions within the electrospray ionization (ESI) interface. These two ions vary only by the site of analyte protonation. Since they are isobaric, they are equally transmitted through the first quadrupole, but are differently fragmented in the collision chamber. The existence of two isobaric ions was deduced by statistical data and the observation of a doubly charged analyte ion. It was hypothesized that the combined presence of [M+H](+) and [M+2H](2+) implies the existence of two different singly charged ion species differing only by the site of protonation. Low- and high-energy interface-induced fragmentation was performed on the samples. The surviving precursor ion population was mass selected and again fragmented in the collision chamber. Equal product ion spectra would be expected. However, very different product ion spectra were observed for the two interface regimes. This is consistent with the assumption that the two postulated isobaric precursor ions show different stability in the interface. Hence the abundance ratio among the two types of surviving precursor ions will shift and change the resulting product ion spectra. The existence of the postulated singly charged ions with multiple chargeable sites was finally confirmed by successful ion mobility separation.
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http://dx.doi.org/10.1002/rcm.3959DOI Listing
April 2009

Studies of peptide a- and b-type fragment ions using stable isotope labeling and integrated ion mobility/tandem mass spectrometry.

J Am Soc Mass Spectrom 2008 Dec 30;19(12):1781-7. Epub 2008 Sep 30.

Michael Barber Centre for Mass Spectrometry, School of Chemistry and Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom.

The structures of peptide a- and b-type fragment ions were studied using synthetic peptides including a set of isomeric peptides, differing in the sequence location of an alanine residue labeled with (15)N and uniformly with (13)C. The pattern of isotope labeling of second-generation fragment ions derived via a(n) and b(n) ions (where n = 4 or 5) suggested that these intermediates existed in part as macrocyclic structures, where alternative sites of ring opening gave rise to different linear forms whose simple cleavage might give rise to the observed final products. Similar conclusions were derived from combined ion mobility/tandem MS analyses where different fragmentation patterns were observed for isomeric a- or b-type ions that display different ion mobilities. These analyses were facilitated by a new approach to the processing of ion mobility/tandem MS data, from which distinct and separate product ion spectra are derived from ions that are incompletely separated by ion mobility. Finally, an example is provided of evidence for a macrocyclic structure for b(n) ions where n = 8 or 9.
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http://dx.doi.org/10.1016/j.jasms.2008.09.024DOI Listing
December 2008