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

   https://doi.org/10.1002/rcm.9192  

   Liu, Fanny_C; Ridgeway, Mark_E; Winfred, J_S_Raaj_Vellore; Polfer, Nicolas_C; Lee, Jusung; Theisen, Alina; Wootton, Christopher_A; Park, Melvin_A; Bleiholder, Christian (September 2021, Rapid Communications in Mass Spectrometry)

   RationaleTandem‐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. MethodsHere, 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. ResultsWe 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. ConclusionsThe 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. 

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2. Tandem trapped ion mobility spectrometry

   https://doi.org/10.1039/c7an02054f  

   Liu, Fanny C.; Ridgeway, Mark E.; Park, Melvin A.; Bleiholder, Christian (January 2018, The Analyst)

   There is currently a strong interest in the use of ion mobility spectrometry-mass spectrometry (IMS-MS) instrumentation for structural biology. In these applications, momentum transfer cross sections derived from IMS-MS measurements are used to reconstruct the three-dimensional analyte structure. Recent reports indicate that additional structural information can be extracted from measuring changes in cross sections in response to changes of the analyte structure. To further this approach, we constructed a tandem trapped IMS analyser (TIMS-TIMS) and incorporated it in a QqTOF mass spectrometer. TIMS-TIMS is constructed by coupling two TIMS analysers via an “interface region” composed of two apertures. We show that peptide oligomers (bradykinin) and native-like protein (ubiquitin) ions can be preserved through the course of an experiment in a TIMS-TIMS analyser. We demonstrate the ability to collisionally-activate as well as to trap mobility-selected ions, followed by subsequent mobility-analysis. In addition to inducing conformational changes, we show that we can fragment low charge states of ubiquitin at >1 mbar between the TIMS analysers with significant sequence coverage. Many fragment ions exhibit multiple features in their TIMS spectra, which means that they may not generally exist as the most stable isomer. The ability of TIMS-TIMS to dissociate mobility-selected protein ions and to measure the cross sections of their fragment ions opens new possibilities for IMS-based structure elucidation. 

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3. Resolving Isomers of Star-Branched Poly(Ethylene Glycols) by IMS-MS Using Multiply Charged Ions

   https://doi.org/10.1021/jasms.0c00045  

   Austin, Calvin A.; Inutan, Ellen D.; Bohrer, Brian C.; Li, Jing; Fischer, Joshua L.; Wijerathne, Kanchana; Foley, Casey D.; Lietz, Christopher B.; Woodall, Daniel W.; Imperial, Lorelie F.; et al (January 2020, Journal of the American Society for Mass Spectrometry)

   null (Ed.)

   Ion mobility spectrometry (IMS) mass spectrometry (MS) centers on the ability to separate gaseous structures by size, charge, shape, and followed by mass-to-charge (m/z). For oligomeric structures, improved separation is hypothesized to be related to the ability to extend structures through repulsive forces between cations electrostatically bonded to the oligomers. Here we show the ability to separate differently branched multiply charged ions of star-branched poly(ethylene glycol) oligomers (up to 2000 Da) regardless of whether formed by electrospray ionization (ESI) charged solution droplets or from charged solid particles produced directly from a surface by matrix-assisted ionization. Detailed structural characterization of isomers of the star-branched compositions was first established using a home-built high-resolution ESI IMS-MS instrument. The doubly charged ions have well-resolved drift times, achieving separation of isomers and also allowing differentiation of star-branched versus linear oligomers. An IMS-MS “snapshot” approach allows visualization of architectural dispersity and (im)purity of samples in a straightforward manner. Analyses capabilities are shown for different cations and ionization methods using commercially available traveling wave IMS-MS instruments. Analyses directly from surfaces using the new ionization processes are, because of the multiply charging, not only associated with the benefits of improved gas-phase separations, relative to that of ions produced by matrix-assisted laser desorption/ionization, but also provide the potential for spatially resolved measurements relative to ESI and other ionization methods. 

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4. Peptide Sequence Influence on the Conformational Dynamics and DNA binding of the Intrinsically Disordered AT-Hook 3 Peptide

   https://doi.org/10.1038/s41598-018-28956-z  

   Garabedian, Alyssa; Bolufer, Alexander; Leng, Fenfei; Fernandez-Lima, Francisco (July 2018, Scientific Reports)

   Abstract The intrinsically disordered ATHP3 was studied at native conditions and in complex with DNA using single amino acid substitutions and high-resolution ion mobility spectrometry coupled to mass spectrometry (trapped IMS-MS). Results showed that ATHP3 can exist in multiple conformations at native conditions (at least 10 conformers were separated), with a variety of prolinecis/transorientations, side chain orientations and protonation sites. When in complex with AT rich DNA hairpins, the -RGRP- core is essential for stabilizing the ATHP3: DNA complex. In particular, the arginine in the sixth position plays an important role during binding to AT-rich regions of hairpin DNA, in good agreement with previous NMR and X-ray data. Mobility based correlation matrices are proposed as a way to reveal differences in structural motifs across the peptide mutants based on the conformational space and relative conformer abundance. 

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5. Superior Differential Ion Mobility Spectrometry of Pendular Macromolecules Using Low-Frequency Rectangular Waveforms

   https://doi.org/10.1021/acs.analchem.4c06841  

   Thurman, Hayden A; Gusachenko, Egor; Anderson, Gordon A; Shvartsburg, Alexandre A (April 2025, Analytical Chemistry)

   Ion mobility spectrometry (IMS) can delineate gas-phase ions and probe their geometries. Coupling with electrospray ionization and MS has brought IMS to structural biology, revealing the macromolecular folding and subunit connectivity. However, the orientational averaging of ion–molecule collision cross sections (Ω) in the linear and field asymmetric waveform IMS (FAIMS) diminishes the resolution and structural specificity. In the novel low-field differential (LOD) IMS, a field too weak for ion heating (and thus FAIMS) aligns strong macrodipoles, capturing their magnitudes and directional Ω across the dipole (Ω⊥). However, the bisinusoidal waveforms (from FAIMS) have compromised the resolution, measurement accuracy, and correlation to the ion properties. Large ions amenable to LODIMS have low mobility and diffuse slowly, allowing the waveform frequencies down to ∼10 kHz. The low field and frequency permit generating the ideal rectangular waveforms with a flexible frequency and duty cycle by direct switching (impractical for FAIMS) in a miniature low-power format. This new IMS stage is evaluated for the exemplary large protein albumin (66 kDa) previously studied using the bisinusoidal waveform. The flat voltages and greater form factor initiate the differential IMS effect at lower fields, expand the separation space, and enable the quantification of Ω⊥ values by varying the duty cycle. 

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