Surface-induced dissociation of protein complexes on a cyclic ion mobility spectrometer.

We describe the implementation of a simple three-electrode surface-induced dissociation (SID) cell on a cyclic ion mobility spectrometer (cIMS) and demonstrate the utility of multipass mobility separations for resolving multiple conformations of protein complexes generated during collision-induced and surface-induced unfolding (CIU & SIU) experiments. In addition to CIU and SIU, SID of protein complexes is readily accomplished within the native instrument software and with no additional external power supplies by entering a single SID collision energy, a simplification in user experience compared to prior implementations. A set of cyclic homomeric protein complexes and a heterohexamer with known CID and SID behavior were analyzed to investigate mass and mobility resolution improvements, the latter of which improved by 20-50% (median: 33%) compared to a linear travelling wave device. Multiple passes of intact complexes, or their SID fragments, increased the mobility resolution by an average of 15% per pass, with the racetrack effect being observed after ∼3 or 4 passes, depending on the drift time spread of the analytes. Even with modest improvements to apparent mobility resolving power, multipass experiments were particularly useful for separating conformations produced from CIU and SIU experiments. We illustrate several examples where either (1) multipass experiments revealed multiple overlapping conformations previously unobserved or obscured due to limited mobility resolution, or (2) CIU or SIU conformations that appeared 'native' in a single pass experiment were actually slightly compacted or expanded, with the change only being measurable through multipass experiments. The work conducted here, the first utilization of multipass cyclic ion mobility for CIU, SIU, and SID of protein assemblies and a demonstration of a wholly integrated SIU/SID workflow, paves the way for widespread adoption of SID technology for native mass spectrometry and also improves our understanding of gas-phase protein complex CIU and SIU conformationomes.

Production and analysis of multiply charged negative ions by liquid atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

RATIONALE: Liquid atmospheric pressure matrix-assisted laser desorption/ionisation (AP-MALDI) has been shown to enable the production of electrospray ionisation (ESI)-like multiply charged analyte ions with little sample consumption and long-lasting, robust ion yield for sensitive analysis by mass spectrometry (MS). Previous reports have focused on positive ion production. Here, we report an initial optimisation of liquid AP-MALDI for ESI-like negative ion production and its application to the analysis of peptides/proteins, DNA and lipids. METHODS: The instrumentation employed for this study is identical to that of earlier liquid AP-MALDI MS studies for positive analyte ion production with a simple non-commercial AP ion source that is attached to a Waters Synapt G2-Si mass spectrometer and incorporates a heated ion transfer tube. The preparation of liquid MALDI matrices is similar to positive ion mode analysis but has been adjusted for negative ion mode by changing the chromophore to 3-aminoquinoline and 9-aminoacridine for further improvements. RESULTS: For DNA, liquid AP-MALDI MS analysis benefited from switching to 9-aminoacridine-based MALDI samples and the negative ion mode, increasing the number of charges by up to a factor of 2 and the analyte ion signal intensities by more than 10-fold compared with the positive ion mode. The limit of detection was recorded at around 10 fmol for ATGCAT. For lipids, negative ion mode analysis provided a fully orthogonal set of detected lipids. CONCLUSIONS: Negative ion mode is a sensitive alternative to positive ion mode in liquid AP-MALDI MS analysis. In particular, the analysis of lipids and DNA benefited from the complementarity of the detected lipid species and the vastly greater DNA ion signal intensities in negative ion mode.

Coupling 193 nm Ultraviolet Photodissociation and Ion Mobility for Sequence Characterization of Conformationally-Selected Peptides.

Ultraviolet photodissociation (UVPD) has emerged as a useful technique for characterizing peptide, protein, and protein complex primary and secondary structure. 193 nm UVPD, specifically, enables extensive covalent fragmentation of the peptide backbone without the requirement of a specific side chain chromophore and with no precursor charge state dependence. We have modified a commercial quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer to include 193 nm UVPD following ion mobility. Ion mobility (IM) is a gas-phase separation technique that enables separation of ions by their size, shape, and charge, providing an orthogonal dimension of separation to mass analysis. Following instrument modifications, we characterized the performance of, and information that could be generated from, this new setup using the model peptides substance P, melittin, and insulin chain B. These experiments show extensive fragmentation across the peptide backbone and a variety of ion types as expected from 193 nm UVPD. Additionally, y-2 ions (along with complementary a+2 and b+2 ions) N-terminal to proline were observed. Combining the IM separation and mobility gating capabilities with UVPD, we demonstrate the ability to accomplish both mass- and mobility-selection of bradykinin des-Arg9 and des-Arg1 peptides followed by complete sequence characterization by UVPD. The new capabilities of this modified instrument demonstrate the utility of combining IM with UVPD because isobaric species cannot be independently selected with a traditional quadrupole alone.

Top-Down Characterization of Denatured Proteins and Native Protein Complexes Using Electron Capture Dissociation Implemented within a Modified Ion Mobility-Mass Spectrometer.

Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument's existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.

Initial Protein Unfolding Events in Ubiquitin, Cytochrome c and Myoglobin Are Revealed with the Use of 213 nm UVPD Coupled to IM-MS.

The initial stages of protein unfolding may reflect the stability of the entire fold and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work, we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation; experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins, the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M + 7H]7+, cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions whilst cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles. Graphical Abstract.

Probing the Dissociation of Protein Complexes by Means of Gas-Phase H/D Exchange Mass Spectrometry.

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND3-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ.

The matrix-assisted laser desorption/ionisation in-source decay of peptides using ion mobility enabled quadrupole-time-of-flight mass spectrometry.

RATIONALE: In-source decay (ISD) matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry with a 1,5-diaminonaphthalene (1,5-DAN) matrix is used for the structural characterisation of peptides. However, MALDI spectra are intrinsically complicated by the presence of matrix ions, which interfere with the peptide fragments. This may cause false-positive results or reduced sequence coverage. This paper reports investigations of ISD processes in an intermediate pressure MALDI ion source and a protocol for the removal of interfering ions using ion mobility separation (IMS). METHODS: An intermediate pressure MALDI source of a Q-IMS-Q-TOF instrument (Synapt G2) has been employed for the ISD of selected peptides using a 1,5-DAN matrix. RESULTS: Successful coupling of the MALDI source tuned for ISD experiments using IMS is demonstrated. The IMS made it possible to remove interfering matrix ions effectively from the spectra and thus to increase the confidence of spectral interpretation. Extensive fragment series corresponding to N-Cα bond cleavages were observed under optimised conditions; on the other hand, weaker series of ions caused by peptide bond cleavages were prevalent for default conditions and/or the α-hydroxycinnamic acid matrix. CONCLUSIONS: Ion mobility has been used for the elimination of matrix ions. The technique has been applied to top-down sequencing of non-tryptic peptides, such as the human palmitoylated analogue of prolactin-releasing peptide used in recent obesity studies, and human and insect antimicrobial peptides.

Comprehensive Tandem-Mass-Spectrometry Coverage of Complex Samples Enabled by Data-Set-Dependent Acquisition.

Tandem mass spectrometry (MS/MS) is an invaluable experimental tool for providing analytical data supporting the identification of small molecules and peptides in mass-spectrometry-based "omics" experiments. Data-dependent MS/MS (DDA) is a real-time MS/MS-acquisition strategy that is responsive to the signals detected in a given sample. However, in analysis of even moderately complex samples with state-of-the-art instrumentation, the speed of MS/MS acquisition is insufficient to offer comprehensive MS/MS coverage of all detected molecules. Data-independent approaches (DIA) offer greater MS/MS coverage, typically at the expense of selectivity or sensitivity. This report describes data-set-dependent MS/MS (DsDA), a novel integration of MS1-data processing and target prioritization to enable comprehensive MS/MS sampling during the initial MS-level experiment. This approach is guided by the premise that in omics experiments, individual injections are typically made as part of a larger set of samples, and feedback between data processing and data acquisition can allow approximately real-time optimization of MS/MS-acquisition parameters and nearly complete MS/MS-sampling coverage. Using a combination of R, Proteowizard, XCMS, and WRENS software, this concept was implemented on a liquid-chromatograph-coupled quadrupole time-of-flight mass spectrometer. The results illustrate comprehensive MS/MS coverage for a set of complex small-molecule samples and demonstrate a strong improvement on traditional DDA.

UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium levels at single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation for increasing the spatial resolution of the HDX-MS experiment, capable of yielding high fragmentation efficiency, high fragment ion diversity, and low precursor ion charge-state dependency.

Tandem Mass Spectrometry in Combination with Product Ion Mobility for the Identification of Phospholipids.

Concerted tandem and traveling wave ion mobility mass spectrometry (CTS analysis) is a unique method that results in a four-dimensional data set including nominal precursor ion mass, product ion mobility, accurate mass of product ion, and ion abundance. This nontargeted lipidomics CTS approach was applied in both positive- and negative-ion mode to phospholipids present in human serum, and the data set was used to evaluate the value of product ion mobility in identifying lipids in a complex mixture. It was determined that the combination of diagnostic product ions and unique collisional cross-section values of product ions is a powerful tool in the structural identification of lipids in a complex biological sample.

Electron Transfer Dissociation of All Ions at All Times, MSETD, in a Quadrupole Time-of-Flight (Q-ToF) Mass Spectrometer.

Data-independent mass spectral acquisition is particularly powerful when combined with ultra-performance liquid chromatography (LC) that provides excellent separation of most components present in a given sample. Data-independent analysis (DIA) consists of alternating full MS scans and scans with fragmentation of all ions within a selected m/z range, providing precursor masses and structure information, respectively. Fragmentation spectra are acquired either by sequential isolation and fragmentation of sliding m/z ranges or fragmenting all ions entering the MS instrument with no ion isolation, termed broadband DIA. Previously, broadband DIA has only been possible using collision induced dissociation (CID). Here, we report the use of electron transfer dissociation (ETD) as the fragmentation technique in broadband DIA instead of traditional collision induced dissociation (CID) during MSE. In this approach, which we refer to as MSETD, we implement the inherent benefits provided by ETD, such as discrimination of leucine and isoleucine, in a DIA setup. The combination of DIA analysis and ETD fragmentation with supplemental CID energy provides a powerful platform to obtain information on all precursors and their sequence from a single experiment. Graphical Abstract ᅟ.

Use of Ultraviolet Photodissociation Coupled with Ion Mobility Mass Spectrometry To Determine Structure and Sequence from Drift Time Selected Peptides and Proteins.

We demonstrate the capabilities of a laser-coupled ion mobility mass spectrometer for analysis of peptide sequence and structure showing ultraviolet photodissociation (UVPD) spectra of mass and mobility selected ions. A Synapt G2-S mass spectrometer has been modified to allow photointeraction of ions post the mobility cell. For this work, we have employed a single wavelength laser, which irradiates at 266 nm. We present the unique capabilities of this instrument and demonstrate several key features. Irradiation of luteinizing hormone releasing hormone (LHRH), growth hormone releasing hexapeptide (GHRP-6), and TrpCage (sequence NLYIQWLKDGGPSSGRPPPS) yields extensive b- and y-type fragmentation as well as a- and c-type ions. In addition, we observe side chain losses, including the indole group from tryptophan, and immonium ions. For negatively charged ions, we show the advantage of using collision-induced dissociation (CID) post-UVPD: radical ions are produced following irradiation, and these fragment with higher efficiency. Further, we have incorporated ion mobility and subsequent drift time gating into the UVPD method allowing the separate analysis of m/z-coincident species, both conformers and multimers. To demonstrate, we selectively dissociate the singly charged dimer or doubly charged monomer of the peptide gramicidin A and conformers of the [M + 5H]5+ form of the peptide melittin. Each mobility selected form has a different "fingerprint" dissociation spectrum, both predominantly containing b and y fragments. Differences in the intensities of various loss channels between the two species were revealed. The smaller conformer of melittin has fewer cleavage sites along the peptide backbone than the larger conformer suggesting considerable structural differences. For gramicidin, a single laser shot UVPD discriminates between primary photodissociation and subsequent fragmentation of fragments. We also show how this modified instrument facilitates activated electron photodissociation. UVPD-IM-MS analysis serves both as a method for peptide sequencing for peptides of similar (or identical) m/z and a method for optical analysis of mobility separated species.

Tuning a High Transmission Ion Guide to Prevent Gas-Phase Proton Exchange During H/D Exchange MS Analysis.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for protein structural analysis has been adopted for many purposes, including biopharmaceutical development. One of the benefits of examining amide proton exchange by mass spectrometry is that it can readily resolve different exchange regimes, as evidenced by either binomial or bimodal isotope patterns. By careful analysis of the isotope pattern during exchange, more insight can be obtained on protein behavior in solution. However, one must be sure that any observed bimodal isotope patterns are not artifacts of analysis and are reflective of the true behavior in solution. Sample carryover and certain stationary phases are known as potential sources of bimodal artifacts. Here, we describe an additional undocumented source of deuterium loss resulting in artificial bimodal patterns for certain highly charged peptides. We demonstrate that this phenomenon is predominantly due to gas-phase proton exchange between peptides and bulk solvent within the initial stages of high-transmission conjoined ion guides. Minor adjustments of the ion guide settings, as reported here, eliminate the phenomenon without sacrificing signal intensity. Such gas-phase deuterium loss should be appreciated for all HDX-MS studies using such ion optics, even for routine studies not focused on interpreting bimodal spectra. Graphical Abstract ᅟ.

Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry.

Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub-millisecond time span after electrospray ionization by ND3 gas can provide structural insights into protein conformers present in solution. Here, we have explored the use of gas-phase HDX-MS for probing the higher-order structure and binding interfaces of protein complexes originating from native solution conditions. Lysozyme ions bound by an oligosaccharide incorporated less deuterium than the unbound ion. Similarly, trypsin ions showed reduced deuterium uptake when bound by the peptide ligand vasopressin. Our results are in good agreement with crystal structures of the native protein complexes, and illustrate that gas-phase HDX-MS can provide a sensitive and simple approach to measure the number of heteroatom-bound non-amide side-chain hydrogens involved in the binding interface of biologically relevant protein complexes.

Extended Gas-Phase Trapping Followed by Surface-Induced Dissociation of Noncovalent Protein Complexes.

Mass spectrometry has emerged as a useful tool in the study of proteins and protein complexes. It is of fundamental interest to explore how the structures of proteins and protein complexes are affected by the absence of solvent and how this alters with increasing time in the gas phase. Here we demonstrate that a range of protein and protein complexes can be confined within the Trap T-wave region of a modified Waters Synapt G2S instrument, including monomeric (ß-lactoglobulin), dimeric (ß-lactoglobulin and enolase), tetrameric (streptavidin, concanavalin A, and pyruvate kinase), and pentameric (C-reactive protein) complexes, ranging in size up to 237 kDa. We demonstrate that complexes can be confined within the Trap region for varying lengths of time over the range 1-60 s and with up to 86% trapping efficiency for 1 s trapping. Furthermore, using model systems, we show that these noncovalent complexes can also be fragmented by surface-induced dissociation (SID) following trapping. SID reveals similar dissociation patterns over all trapping times studied for unactivated protein complexes, suggesting that any conformational changes occurring over this time scale are insufficient to cause substantial differences in the SID spectra of these complexes. Intentional alteration of structure by cone activation produces a distinct SID spectrum, with the differences observed being conserved, in comparison to unactivated complex, after trapping. However, subtle differences in the SID spectra of the activated complex are also observed as a function of trapping time.

Characterization of top-down ETD in a travelling-wave ion guide.

Top-down sequencing methods are becoming increasingly relevant for protein characterization, in particular electron capture (ECD) and electron transfer dissociation (ETD) which allow for extensive backbone cleavage with minimal side reactions. The ability to obtain sequence-specific fragments while maintaining aspects of the higher-order structure, as well as the position of deuterium labels in H/D exchange, has attracted interest from scientists in the field of structural proteomics. Recently, ETD has also been combined with ion mobility on commercially available quadrupole/time-of-flight instruments, and this implementation paves the way to novel structural studies and investigation of the ETD process itself. In the current work, we investigate the use of ETD for fragmentation of standard peptides and proteins and provide a detailed description of the effect of the parameters controlling the time and efficiency of the reaction. We also highlight how the combination with ion mobility separation after electron transfer provides extended analytical benefits, such as assignment of fragments to a specific charge-reduced state of the precursor.

Extensive Charge Reduction and Dissociation of Intact Protein Complexes Following Electron Transfer on a Quadrupole-Ion Mobility-Time-of-Flight MS.

Non-dissociative charge reduction, typically considered to be an unwanted side reaction in electron transfer dissociation (ETD) experiments, can be enhanced significantly in order to reduce the charge state of intact protein complexes to as low as 1+ on a commercially available Q-IM-TOF instrument. This allows for the detection of large complexes beyond 100,000 m/z, while at the same time generating top-down ETD fragments, which provide sequence information from surface-exposed parts of the folded structure. Optimization of the supplemental activation has proven to be crucial in these experiments and the charge-reduced species are most likely the product of both proton transfer (PTR) and non-dissociative electron transfer (ETnoD) reactions that occur prior to the ion mobility cell. Applications of this approach range from deconvolution of complex spectra to the manipulation of charge states of gas-phase ions.

Changes in protein structure monitored by use of gas-phase hydrogen/deuterium exchange.

The study of protein conformation by solution-phase hydrogen/deuterium exchange (HDX) coupled to MS is well documented. This involves monitoring the exchange of backbone amide protons with deuterium and provides details concerning the protein's tertiary structure. However, undesired back-exchange during post-HDX analyses can be difficult to control. Here, gas-phase HDX-MS, during which labile hydrogens on amino acid side chains are exchanged in sub-millisecond time scales, has been employed to probe changes within protein structures. Addition of the solvent 2,2,2-trifluoroethanol to a protein in solution can affect the structure of the protein, resulting in an increase in secondary and/or tertiary structure which is detected using circular dichroism. Using a Synapt G2-S ESI-mass spectrometer modified to allow deuterated ammonia into the transfer ion guide (situated between the ion mobility cell and the TOF analyser), gas-phase HDX-MS is shown to reflect minor structural changes experienced by the proteins ß-lactoglobulin and ubiquitin, as observed by the reduction in the level of deuterium incorporation. Additionally, the use of gas-phase HDX-MS to distinguish between co-populated proteins conformers within a solution is demonstrated with the disordered protein calmodulin; the gas-phase HDX-MS results correspond directly with complementary data obtained by use of ion mobility spectrometry-MS.

Simple setup for gas-phase H/D exchange mass spectrometry coupled to electron transfer dissociation and ion mobility for analysis of polypeptide structure on a liquid chromatographic time scale.

Gas-phase hydrogen/deuterium exchange (HDX) is a fast and sensitive, yet unharnessed analytical approach for providing information on the structural properties of biomolecules, in a complementary manner to mass analysis. Here, we describe a simple setup for ND3-mediated millisecond gas-phase HDX inside a mass spectrometer immediately after ESI (gas-phase HDX-MS) and show utility for studying the primary and higher-order structure of peptides and proteins. HDX was achieved by passing N2-gas through a container filled with aqueous deuterated ammonia reagent (ND3/D2O) and admitting the saturated gas immediately upstream or downstream of the primary skimmer cone. The approach was implemented on three commercially available mass spectrometers and required no or minor fully reversible reconfiguration of gas-inlets of the ion source. Results from gas-phase HDX-MS of peptides using the aqueous ND3/D2O as HDX reagent indicate that labeling is facilitated exclusively through gaseous ND3, yielding similar results to the infusion of purified ND3-gas, while circumventing the complications associated with the use of hazardous purified gases. Comparison of the solution-phase- and gas-phase deuterium uptake of Leu-Enkephalin and Glu-Fibrinopeptide B, confirmed that this gas-phase HDX-MS approach allows for labeling of sites (heteroatom-bound non-amide hydrogens located on side-chains, N-terminus and C-terminus) not accessed by classical solution-phase HDX-MS. The simple setup is compatible with liquid chromatography and a chip-based automated nanoESI interface, allowing for online gas-phase HDX-MS analysis of peptides and proteins separated on a liquid chromatographic time scale at increased throughput. Furthermore, online gas-phase HDX-MS could be performed in tandem with ion mobility separation or electron transfer dissociation, thus enabling multiple orthogonal analyses of the structural properties of peptides and proteins in a single automated LC-MS workflow.

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

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).