High Resolution Trapped Ion Mobility Spectrometery of Peptides

In the present work, we employ trapped ion mobility spectrometry (TIMS) for conformational analysis of several model peptides. The TIMS distributions are extensively compared to recent ion mobility spectrometry (IMS) studies reported in the literature. At a resolving power (<i>R</i>) exceeding 250, many new features, otherwise hidden by lower resolution IMS analyzers, are revealed. Though still principally limited by the plurality of conformational states, at present, TIMS offers <i>R</i> up to ∼3 to 8 times greater than modern drift tube or traveling wave IMS techniques, respectively. Unlike differential IMS, TIMS not only is able to resolve congested conformational features but also can be used to determine information about their relative size, via the ion-neutral collision cross section, offering a powerful new platform to probe the structure and dynamics of biochemical systems in the gas phase

Targeted High-Resolution Ion Mobility Separation Coupled to Ultrahigh-Resolution Mass Spectrometry of Endocrine Disruptors in Complex Mixtures

Traditional separation and detection of targeted compounds from complex mixtures from environmental matrices requires the use of lengthy prefractionation steps and high-resolution mass analyzers due to the large number of chemical components and their large structural diversity (highly isomeric). In the present work, selected accumulation trapped ion mobility spectrometry (SA-TIMS) is coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) for direct separation and characterization of targeted endocrine-disrupting compounds (EDC) from a complex environmental matrix in a single analysis. In particular, targeted identification based on high-resolution mobility (<i>R</i> ∼ 70–120) and ultrahigh-resolution mass measurements (<i>R</i> > 400 000) of seven commonly targeted EDC and their isobars (e.g., bisphenol A, (<i>Z</i>)- and (<i>E</i>)-diethylstilbestrol, hexestrol, estrone, α-estradiol, and 17-ethynylestradiol) is shown from a complex mixture of water-soluble organic matter (e.g., Suwannee River Fulvic Acid Standard II) complemented with reference standard measurements and theoretical calculations (<3% error)

Structural Analysis of the Glycoprotein Complex Avidin by Tandem-Trapped Ion Mobility SpectrometryMass 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

Isomerization Kinetics of AT Hook Decapeptide Solution Structures

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

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%

Isomerization Kinetics of AT Hook Decapeptide Solution Structures

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

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

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

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

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

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

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

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