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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)

   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.

   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.

   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.

   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.

    

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2. Metal ions induced secondary structure rearrangements: mechanically interlocked lasso vs. unthreaded branched-cyclic topoisomers

   https://doi.org/10.1039/c8an00138c  

   Jeanne Dit Fouque, Kevin ; Moreno, Javier ; Hegemann, Julian D. ; Zirah, Séverine ; Rebuffat, Sylvie ; Fernandez-Lima, Francisco ( January 2018 , The Analyst)

   Metal ions can play a significant role in a variety of important functions in protein systems including cofactor for catalysis, protein folding, assembly, structural stability and conformational change. In the present work, we examined the influence of alkali (Na, K and Cs), alkaline earth (Mg and Ca) and transition (Co, Ni and Zn) metal ions on the conformational space and analytical separation of mechanically interlocked lasso peptides. Syanodin I, sphingonodin I, caulonodin III and microcin J25, selected as models of lasso peptides, and their respective branched-cyclic topoisomers were submitted to native nESI trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high mobility resolving power of TIMS permitted to group conformational families regardless of the metal ion. The lower diversity of conformational families for syanodin I as compared to the other lasso peptides supports that syanodin I probably forms tighter binding interactions with metal ions limiting their conformational space in the gas-phase. Conversely, the higher diversity of conformational families for the branched-cyclic topologies further supports that the metal ions probably interact with a higher number of electronegative groups arising from the fully unconstraint C-terminal part. A correlation between the lengths of the loop and the C-terminal tail with the conformational space of lasso peptides becomes apparent upon addition of metal ions. It was shown that the threaded C-terminal region in lasso peptides allows only for distinct interactions of the metal ion with either residues in the loop or tail region. This limits the size of the interacting region and apparently leads to a bias of metal ion binding in either the loop or tail region, depending whichever section is larger in the respective lasso peptide. For branched-cyclic peptides, the non-restricted C-terminal tail allows metal coordination by residues throughout this region, which can result in gas-phase structures that are sometimes even more compact than the lasso peptides. The high TIMS resolution also resulted in the separation of almost all lasso and branched-cyclic topoisomer metal ions ( r ∼ 2.1 on average). It is also shown that the metal incorporation ( e.g. , doubly cesiated species) can lead to the formation of a simplified IMS pattern (or preferential conformers), which results in baseline analytical separation and discrimination between lasso and branched-cyclic topologies using TIMS-MS. 

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3. Comparison of gaseous ubiquitin ion structures obtained from a solid and solution matrix using ion mobility spectrometry/mass spectrometry

   https://doi.org/10.1002/rcm.8793  

   Inutan, Ellen D. ; Jarois, Dean R. ; Lietz, Christopher B. ; El‐Baba, Tarick J. ; Elia, Efstathios A. ; Karki, Santosh ; Sampat, Andjoe A. S. ; Foley, Casey D. ; Clemmer, David E. ; Trimpin, Sarah ( August 2020 , Rapid Communications in Mass Spectrometry)

   Examining surface protein conformations, and especially achieving this with spatial resolution, is an important goal. The recently discovered ionization processes offer spatial‐resolution measurements similar to matrix‐assisted laser desorption/ionization (MALDI) and produce charge states similar to electrospray ionization (ESI) extending higher‐mass protein applications directly from surfaces on high‐performance mass spectrometers. Studying a well‐interrogated protein by ion mobility spectrometry‐mass spectrometry (IMS‐MS) to access effects on structures using a solidvs.solvent matrix may provide insights.

   Ubiquitin was studied by IMS‐MS using new ionization processes with commercial and homebuilt ion sources and instruments (Waters SYNAPT G2(S)) and homebuilt 2 m drift‐tube instrument; MS™ sources). Mass‐to‐charge and drift‐time (t_d)‐measurements are compared for ubiquitin ions obtained byinletandvacuumionization using laserspray ionization (LSI), matrix‐ (MAI) and solvent‐assisted ionization (SAI), respectively, and compared with those from ESI under conditions that are most comparable.

   Using the same solution conditions with SYNAPT G2(S) instruments, t_d‐distributions of various ubiquitin charge states from MAI, LSI, and SAI are similar to those from ESI using a variety of solvents, matrices, extraction voltages, a laser, and temperature only, showing subtle differences in more compact features within the elongated distribution of structures. However, on a homebuilt drift‐tube instrument, within the elongated distribution of structures, both similar and different t_d‐distributions are observed for ubiquitin ions obtained by MAI and ESI. MAI‐generated ions are frequently narrower in their t_d‐distributions.

   Direct comparisons between ESI and the new ionization methods operational directly from surfaces suggest that the protein in its solution structure prior to exposure to the ionization event is either captured (frozen out) at the time of crystallization, or that the protein in the solid matrix is associated with sufficient solvent to maintain the solution structure, or, alternatively, that the observed structures are those related to what occurs in the gas phase with ESI‐ or MAI‐generated ions and not with the solution structures.

    

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4. Imaging mass spectrometry reveals complex lipid distributions across Staphylococcus aureus biofilm layers

   https://doi.org/10.1016/j.jmsacl.2022.09.003  

   Rivera, Emilio S. ; Weiss, Andy ; Migas, Lukasz G. ; Freiberg, Jeffrey A. ; Djambazova, Katerina V. ; Neumann, Elizabeth K. ; Van de Plas, Raf ; Spraggins, Jeffrey M. ; Skaar, Eric P. ; Caprioli, Richard M. ( November 2022 , Journal of Mass Spectrometry and Advances in the Clinical Lab)

   Introduction Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. Methods S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. Results Implementation of TIMS led to a ∼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) – CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously. Conclusions High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies. 

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5. Detection of firearm discharge residue from skin swabs using trapped ion mobility spectrometry coupled to mass spectrometry

   https://doi.org/10.1039/c8ay00658j  

   McKenzie-Coe, Alan ; Bell, Suzanne ; Fernandez-Lima, Francisco ( January 2018 , Analytical Methods)

   In the present work, a novel workflow for the detection of both elemental and organic constituents of the firearm discharge residue from skin swabs was developed using trapped ion mobility spectrometry coupled to mass spectrometry. The small sample size (<10 μL), high specificity and short analysis time (few min) permits the detection of inorganic residues (IGSR; inorganic gunshot residues) and organic residues (OGSR) from one sample and in a single analysis. The analytical method is based on the simultaneous extraction of inorganic and organic species assisted by the formation organometallic complexes ( e.g. , 15–5 crown ethers for the sequestering of metals and nitrate species), followed by fast, post-ionization, high resolution mobility ( R IMS ∼ 150–250) and mass separations ( R MS ∼ 20–40k) with isotopic pattern recognition. The analytical performance is illustrated as a proof of concept for the case of the simultaneous detection of Ba +2 , Pb +2 , Cu + , K + , NO 3 − , diphenylamine (DPA), ethyl centralite (EC) and 2,4 dinitrotoluene (DNT) in positive and negative nESI-TIMS-MS modes. Candidate structures are proposed and collisional cross sections are reported for all organic and organometallic species of interest. 

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