Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Top/Middle-Down Characterization of α-Synuclein Glycoforms.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

Top-Down Protein Analysis by Tandem-Trapped Ion Mobility Spectrometry/Mass Spectrometry (Tandem-TIMS/MS) Coupled with Ultraviolet Photodissociation (UVPD) and Parallel Accumulation/Serial Fragmentation (PASEF) MS/MS Analysis.

"Top-down" proteomics analyzes intact proteins and identifies proteoforms by their intact mass as well as the observed fragmentation pattern in tandem mass spectrometry (MS/MS) experiments. Recently, hybrid ion mobility spectrometry-mass spectrometry (IM/MS) methods have gained traction for top-down experiments, either by allowing top-down analysis of individual isomers or alternatively by improving signal/noise and dynamic range for fragment ion assignment. We recently described the construction of a tandem-trapped ion mobility spectrometer/mass spectrometer (tandem-TIMS/MS) coupled with an ultraviolet (UV) laser and demonstrated a proof-of-principle for top-down analysis by UV photodissociation (UVPD) at 2-3 mbar. The present work builds on this with an exploration of a top-down method that couples tandem-TIMS/MS with UVPD and parallel-accumulation serial fragmentation (PASEF) MS/MS analysis. We first survey types and structures of UVPD-specific fragment ions generated in the 2-3 mbar pressure regime of our instrument. Notably, we observe UVPD-induced fragment ions with multiple conformations that differ from those produced in the absence of UV irradiation. Subsequently, we discuss how MS/MS spectra of top-down fragment ions lend themselves ideally for probability-based scoring methods developed in the bottom-up proteomics field and how the ability to record automated PASEF-MS/MS spectra resolves ambiguities in the assignment of top-down fragment ions. Finally, we describe the coupling of tandem-TIMS/MS workflows with UVPD and PASEF-MS/MS analysis for native top-down protein analysis.

Top-"Double-Down" Mass Spectrometry of Histone H4 Proteoforms: Tandem Ultraviolet-Photon and Mobility/Mass-Selected Electron Capture Dissociations.

Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approach─ultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)─is illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (∼75%) with significant faster data acquisition in the ToF MS/MS platform (∼3 vs ∼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (∼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c305+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me2, 2Ac + Me2 and 3Ac + Me2), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c305+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.

Trapped Ion Mobility Spectrometry, Ultraviolet Photodissociation, and Time-of-Flight Mass Spectrometry for Gas-Phase Peptide Isobars/Isomers/Conformers Discrimination.

Trapped ion mobility spectrometry (TIMS) when coupled with mass spectrometry (MS) offers great advantages for the separation of isobaric, isomeric, and/or conformeric species. In the present work, we report the advantages of coupling TIMS with a low-cost, ultraviolet photodissociation (UVPD) linear ion trap operated at few mbars prior to time-of-flight (ToF) MS analysis for the effective characterization of isobaric, isomeric, and/or conformeric species based on mobility-selected fragmentation patterns. These three traditional challenges to MS-based separations are illustrated for the case of biologically relevant model systems: H3.1 histone tail PTM isobars (K4Me3/K18Ac), lanthipeptide regioisomers (overlapping/nonoverlapping ring patterns), and a model peptide conformer (angiotensin I). The sequential nature of the TIMS operation allows for effective synchronization with the ToF MS scans, in addition to parallel operation between the TIMS and the UVPD trap. Inspection of the mobility-selected UVPD MS spectra showed that for all three cases considered, unique fragmentation patterns (fingerprints) were observed per mobility band. Different from other IMS-UVPD implementations, the higher resolution of the TIMS device allowed for high mobility resolving power (R > 100) and effective mobility separation. The mobility selected UVPD MS provided high sequence coverage (>85%) with a fragmentation efficiency up to ∼40%.

Tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS): a promising analytical method for investigating heterogenous samples.

Ion mobility spectrometry/mass spectrometry (IMS/MS) is widely used to study various levels of protein structure. Here, we review the current state of affairs in tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS). Two different tTIMS/MS instruments are discussed in detail: the first tTIMS/MS instrument, constructed from coaxially aligning two TIMS devices; and an orthogonal tTIMS/MS configuration that comprises an ion trap for irradiation of ions with UV photons. We discuss the various workflows the two tTIMS/MS setups offer and how these can be used to study primary, tertiary, and quaternary structures of protein systems. We also discuss, from a more fundamental perspective, the processes that lead to denaturation of protein systems in tTIMS/MS and how to soften the measurement so that biologically meaningful structures can be characterised with tTIMS/MS. We emphasize the concepts underlying tTIMS/MS to underscore the opportunities tandem-ion mobility spectrometry methods offer for investigating heterogeneous samples.

Tandem-trapped ion mobility spectrometry/mass spectrometry coupled with ultraviolet photodissociation.

RATIONALE: Tandem-ion mobility spectrometry/mass spectrometry methods have recently gained traction for the structural characterization of proteins and protein complexes. However, ion activation techniques currently coupled with tandem-ion mobility spectrometry/mass spectrometry methods are limited in their ability to characterize structures of proteins and protein complexes. METHODS: Here, we describe the coupling of the separation capabilities of tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS) with the dissociation capabilities of ultraviolet photodissociation (UVPD) for protein structure analysis. RESULTS: We establish the feasibility of dissociating intact proteins by UV irradiation at 213 nm between the two TIMS devices in tTIMS/MS and at pressure conditions compatible with ion mobility spectrometry (2-3 mbar). We validate that the fragments produced by UVPD under these conditions result from a radical-based mechanism in accordance with prior literature on UVPD. The data suggest stabilization of fragment ions produced from UVPD by collisional cooling due to the elevated pressures used here ("UVnoD2"), which otherwise do not survive to detection. The data account for a sequence coverage for the protein ubiquitin comparable to recent reports, demonstrating the analytical utility of our instrument in mobility-separating fragment ions produced from UVPD. CONCLUSIONS: The data demonstrate that UVPD carried out at elevated pressures of 2-3 mbar yields extensive fragment ions rich in information about the protein and that their exhaustive analysis requires IMS separation post-UVPD. Therefore, because UVPD and tTIMS/MS each have been shown to be valuable techniques on their own merit in proteomics, our contribution here underscores the potential of combining tTIMS/MS with UVPD for structural proteomics.

Implications of Blanc's Law for Use in Trapped Ion Mobility Spectrometry.

Blanc's Law has served as a way to predict the mobilities of ions in mixed drift gases for over 100 years yet has remained largely unexplored using newer ion mobility spectrometry (IMS) configurations, including traveling wave and trapped IMS (TIMS) systems. Here, we evaluate a drift-tube IMS (DTIMS) and compare it to a similar set of experiments performed in TIMS. We found that Blanc's Law can be applied in a DTIMS to determine the mobility of an analyte in the minor gas component of a ternary mixed drift gas system within 2% error. Additionally, the calibration procedure for TIMS to convert elution voltages into a mobility value corrects for significant deviations (>4%) from Blanc's Law in the elution voltage domain. For the range of gas identities probed in this effort, up to an 11% error in calibrated mobilities was observed when using a gas mixture in the TIMS that differed from the gas used for the reference mobility. However, when the gas mixture within the TIMS was the same as the respective calibrant mobilities, calibration errors within the TIMS were as low as 0.01%. Interestingly, when probing the behavior of ions with argon-containing mixtures within the TIMS, the current accepted paradigm of elution voltage being proportional to inverse mobilities in TIMS calibrations procedures was shown to deviate substantially from the trends observed with DTIMS measurements. With this initial effort, foundations for future mixed drift gas measurements in TIMS are set for expanded analyte classes and larger molecules.

Surface-Induced Dissociation of Protein Complexes Selected by Trapped Ion Mobility Spectrometry.

Native mass spectrometry (nMS), particularly in conjunction with gas-phase ion mobility spectrometry measurements, has proven useful as a structural biology tool for evaluating the stoichiometry, conformation, and topology of protein complexes. Here, we demonstrate the combination of trapped ion mobility spectrometry (TIMS) and surface-induced dissociation (SID) on a Bruker SolariX XR 15 T FT-ICR mass spectrometer for the structural analysis of protein complexes. We successfully performed SID on mobility-selected protein complexes, including the streptavidin tetramer and cholera toxin B with bound ligands. Additionally, TIMS-SID was employed on a mixture of the peptides desArg1 and desArg9 bradykinin to mobility-separate and identify the individual peptides. Importantly, results show that native-like conformations can be maintained throughout the TIMS analysis. The TIMS-SID spectra are analogous to SID spectra acquired using quadrupole mass selection, indicating little measurable, if any, structural rearrangement during mobility selection. Mobility parking was used on the ion or mobility of interest and 50-200 SID mass spectra were averaged. High-quality TIMS-SID spectra were acquired over a period of 2-10 min, comparable to or slightly longer than SID coupled with ion mobility on various instrument platforms in our laboratory. The ultrahigh resolving power of the 15 T FT-ICR allowed for the identification and relative quantification of overlapping SID fragments with the same nominal m/z based on isotope patterns, and it shows promise as a platform to probe small mass differences, such as protein/ligand binding or post-translational modifications. These results represent the potential of TIMS-SID-MS for the analysis of both protein complexes and peptides.

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies.

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K0 = 0.185-1.84 cm2·V-1·s-1), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCSN2) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6-73), Tuning Mix oligomers (n = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1-4), carbonic anhydrase, ß clamp (n = 1-4), topoisomerase IB, bovine serum albumin (n = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1-2)) covering a wide mass (up to m/z 19 000) and CCS range (up to 22 000 Å2 with <0.6% relative standard deviation (RSD)).

Accurate Identification of Isomeric Glycans by Trapped Ion Mobility Spectrometry-Electronic Excitation Dissociation Tandem Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) has become a powerful tool for glycan structural characterization due to its ability to separate isomers and provide collision cross section (CCS) values that facilitate structural assignment. However, IM-based isomer analysis may be complicated by the presence of multiple gas-phase conformations of a single structure that not only increases difficulty in isomer separation but can also introduce the possibility for misinterpretation of conformers as isomers. Here, the ion mobility behavior of several sets of isomeric glycans, analyzed as their permethylated derivatives, in both nonreduced and reduced forms, was investigated by gated-trapped ion mobility spectrometry (G-TIMS). Notably, reducing-end reduction, commonly performed to remove anomerism-induced chromatographic peak splitting, did not eliminate the conformational heterogeneity of permethylated glycans in the gas phase. At a mobility resolving power of ∼100, 14 out of 22 structures showed more than one conformation. These results highlight the need to use IMS devices with high mobility resolving power for better separation of isomers and to acquire additional structural information that can differentiate isomers from conformers. Online electronic excitation dissociation (EED) MS/MS analysis of isomeric glycan mixtures following G-TIMS separation showed that EED can generate isomer-specific fragments while producing nearly identical tandem mass spectra for conformers, thus allowing confident identification of isomers with minimal evidence of any ambiguity resulting from the presence of conformers. G-TIMS EED MS/MS analysis of N-linked glycans released from ovalbumin revealed that several mobility features previously thought to arise from isomeric structures were conformers of a single structure. Finally, analysis of ovalbumin N-glycans from different sources showed that the G-TIMS EED MS/MS approach can accurately determine the batch-to-batch variations in glycosylation profiles at the isomer level, with confident assignment of each isomeric structure.

Identification of Phosphatidylcholine Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions.

Gas-phase ion/ion reactions have been enabled on a commercial dual source, hybrid QhFT-ICR mass spectrometer for use during imaging mass spectrometry experiments. These reactions allow for the transformation of the ion type most readily generated from the tissue surface to an ion type that gives improved chemical structural information upon tandem mass spectrometry (MS/MS) without manipulating the tissue sample. This process is demonstrated via the charge inversion reaction of phosphatidylcholine (PC) lipid cations generated from rat brain tissue via matrix-assisted laser desorption/ionization (MALDI) with 1,4-phenylenedipropionic acid (PDPA) reagent dianions generated via electrospray ionization (ESI). Collision-induced dissociation (CID) of the resulting demethylated PC product anions allows for the determination of the lipid fatty acyl tail identities and positions, which is not possible via CID of the precursor lipid cations. The abundance of lipid isomers revealed by this workflow is found to vary significantly in different regions of the brain. As each isoform may have a unique cellular function, these results underscore the importance of accurately separating and identifying the many isobaric and isomeric lipids and metabolites that can complicate image interpretation and spectral analysis.

Evaluation of Trapped Ion Mobility Spectrometry Source Conditions Using Benzylammonium Thermometer Ions.

A key aspect of reduced pressure ion mobility spectrometry (IMS) experiments is to identify experimental conditions that minimize the role of collisional energy transfer that allows for assessing effective ion-neutral collision cross sections of metabolites, peptides, and proteins in "native-like" or compact states. Across two separate experimental campaigns using a prototype trapped ion mobility spectrometer (TIMS) coupled to a time-of-flight mass spectrometer, we present independent findings that support the results recently published by Morsa et al. using a different set of thermometer ions (Morsa et al. Anal. Chem. 2020, 92 (6), 4573-4582). First, using five para-substituted benzylammonium ions, we conducted survival yield experiments to assess ion internal energy across different experimental settings. Results from the present set of experiments illustrate that greater ion heating occurs at lower pressures and higher voltage settings applied to the TIMS. At the "softest" settings where the benzylammonium thermometer ions have an effective average energy of 1.73 eV, we observe the majority of bradykinin in the compact state. Under more extreme operating conditions where the energy of the benzylammonium ions varies from 1.83 to 1.86 eV, the bradykinin transitions from the compact to the elongated state. In addition to independently confirming the findings of Morsa et al., we also report the mobilities for the benzylammonium parent and fragment ions using the tandem drift-tube-TIMS calibration procedure described by Naylor et al. ( J. Am. Soc. Mass Spectrom. 2019, 30 (10), 2152-2162).

Structural Analysis of the Glycoprotein Complex Avidin by Tandem-Trapped Ion Mobility Spectrometry-Mass Spectrometry (Tandem-TIMS/MS).

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

Validation of Calibration Parameters for Trapped Ion Mobility Spectrometry.

Using contemporary theory for ion mobility spectrometry (IMS), gas-phase ion mobilities within a trapped ion mobility-mass spectrometer (TIMS) are not easily deduced using first principle equations due to non-linear pressure changes and consequently variations in E/N. It is for this reason that prior literature values have traditionally been used for TIMS calibration. Additionally, given that verified mobility standards currently do not exist and the that the exact conditions used to measure reported literature values may not always represent the environment within the TIMS, a direct approach to validating the behavior of the TIMS system is warranted. A calibration procedure is presented where an ambient pressure, ambient temperature, two-gate, printed circuit board drift-tube IMS (PCBIMS) is coupled to the front of a TIMS allowing reduced mobilities to be directly measured on the same instrument as the TIMS. These measured mobilities were used to evaluate the TIMS calibration procedure which correlates reduced mobility and TIMS elution voltages with literature values. When using the measured PCBIMS-reduced mobilities of tetraalkyl ammonium salts and tune mix for TIMS calibration of the alkyltrimethyl ammonium salts, the percent error is less than 1% as compared with using the reported literature K0 values where the percent error approaches 5%. This method provides a way to obtain accurate reference mobilities for ion mobility techniques that require a calibration step (i.e., TIMS and TWAVE).

Recommendations for reporting ion mobility Mass Spectrometry measurements.

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.

Characterization and Quantification of Highly Sulfated Glycosaminoglycan Isomers by Gated-Trapped Ion Mobility Spectrometry Negative Electron Transfer Dissociation MS/MS.

Glycosaminoglycans (GAGs) play vital roles in many biological processes and are naturally present as complex mixtures of polysaccharides with tremendous structural heterogeneity, including many structural isomers. Mass spectrometric analysis of GAG isomers, in particular highly sulfated heparin (Hep) and heparan sulfate (HS), is challenging because of their structural similarity and facile sulfo losses during analysis. Herein, we show that highly sulfated Hep/HS isomers may be resolved by gated-trapped ion mobility spectrometry (gated-TIMS) with negligible sulfo losses. Subsequent negative electron transfer dissociation (NETD) tandem mass spectrometry (MS/MS) analysis of TIMS-separated Hep/HS isomers generated extensive glycosidic and cross-ring fragments for confident isomer differentiation and structure elucidation. The high mobility resolution and preservation of labile sulfo modifications afforded by gated-TIMS MS analysis also allowed relative quantification of highly sulfated heparin isomers. These results show that the gated-TIMS-NETD MS/MS approach is useful for both qualitative and quantitative analysis of highly sulfated Hep/HS compounds in a manner not possible with other techniques.

The effects of solution additives and gas-phase modifiers on the molecular environment and conformational space of common heme proteins.

RATIONALE: The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas-phase modifiers on biomolecules characterized using ion mobility techniques. METHODS: The effect of solution additives and gas-phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100-500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time-of-flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization. RESULTS: Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas-phase modifiers (i.e., N2 doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas-modifier mass and size and changes in the relative abundances and number of IMS bands are observed. CONCLUSIONS: We attribute the observed changes in the mobility profiles in the presence of gas-phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.

Utilizing Ion Mobility to Identify Isobaric Post-Translational Modifications: Resolving Acrolein and Propionyl Lysine Adducts by TIMS Mass Spectrometry.

Protein post-translational modifications provide critical proteomic details towards elucidating mechanisms of altered protein function due to toxic exposure, altered metabolism, or disease pathogenesis. Lysine propionylation is a recently described modification that occurs due to metabolic alterations in propionyl-CoA metabolism and sirtuin depropionylase activity. Acrolein is a toxic aldehyde generated through exogenous and endogenous pathways, such as industrial exposure, cigarette smoke inhalation, and non-enzymatic lipid peroxidation. Importantly, lysine modifications arising from propionylation and acroleination can be isobaric - indistinguishable by mass spectrometry - and inseparable via reverse-phase chromatography. Here, we present the novel application of trapped ion mobility spectrometry (TIMS) to resolve such competing isobaric lysine modifications. Specifically, the PTM products of a small synthetic peptide were analyzed using a prototype TIMS - time-of-flight mass spectrometer (TIMS-TOF). In that the mobilities of these propionylated and acroleinated peptides differ by only 1%, a high-resolution mobility analysis is required to resolve the two. We were able to achieve more than sufficient resolution in the TIMS analyzer (~170), readily separating these isobars.

Coupling trapped ion mobility spectrometry to mass spectrometry: trapped ion mobility spectrometry-time-of-flight mass spectrometry versus trapped ion mobility spectrometry-Fourier transform ion cyclotron resonance mass spectrometry.

RATIONALE: There is a need for fast, post-ionization separation during the analysis of complex mixtures. In this study, we evaluate the use of a high-resolution mobility analyzer with high-resolution and ultrahigh-resolution mass spectrometry for unsupervised molecular feature detection. Goals include the study of the reproducibility of trapped ion mobility spectrometry (TIMS) across platforms, applicability range, and potential challenges during routine analysis. METHODS: A TIMS analyzer was coupled to time-of-flight mass spectrometry (TOF MS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) instruments for the analysis of singly charged species in the m/z 150-800 range of a complex mixture (Suwannee River Fulvic Acid Standard). Molecular features were detected using an unsupervised algorithm based on chemical formula and IMS profiles. RESULTS: TIMS-TOF MS and TIMS-FT-ICR MS analysis provided 4950 and 7760 m/z signals, 1430 and 3050 formulas using the general Cx Hy N0-3 O0-19 S0-1 composition, and 7600 and 22 350 [m/z; chemical formula; K; CCS] features, respectively. CONCLUSIONS: TIMS coupled to TOF MS and FT-ICR MS showed similar performance and high reproducibility. For the analysis of complex mixtures, both platforms were able to capture the major trends and characteristics; however, as the chemical complexity at the level of nominal mass increases with m/z (m/z >300-350), only TIMS-FT-ICR MS was able to report the lower abundance compositional trends.