Conditions for Analysis of Native Protein Structures Using Uniform Field Drift Tube Ion Mobility Mass Spectrometry and Characterization of Stable Calibrants for TWIM-MS

Harrison, Julian A.; Kelso, Céline; Pukala, Tara L.; Beck, Jennifer L.

doi:10.1007/s13361-018-2074-z

Cited by 30 publications

(37 citation statements)

References 45 publications

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“…The comparison of literature CCS values with those determined from this work showed excellent agreement (see linearity in Figure 4c with R 2 of 0.9989). 15,16,45,46,52 This agreement provides further confidence on the use of TIMS-MS technology for structural biology studies.…”

Section: ■ Results and Discussionmentioning

confidence: 59%

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies

et al. 2021

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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 (K 0 = 0.185−1.84 cm 2 •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 (CCS N 2 ) 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, β 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 Å 2 with <0.6% relative standard deviation (RSD)).

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Section: ■ Results and Discussionmentioning

confidence: 59%

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies

et al. 2021

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“…Literature CCS values for each protein in different charge states are shown in SI Tables S1 and S2. A range of values have been reported, and these can be explained by the use of different buffer gases and experimental conditions, 38,40,69,70 and by the presence of multiple conformers populated for a given charge state in the gas-phase. 38,40 These observations highlight the importance of reporting instrumental parameters that influence the mobility of ions, such as the type of drift gas, temperature, pressure, and electric field, and the method of sample preparation (solvent, any additives and pH) to enable comparison of experimentally derived CCSs between different laboratories.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Probing the Fundamentals of Native Liquid Extraction Surface Analysis Mass Spectrometry of Proteins: Can Proteins Refold during Extraction?

Illes‐Toth

Cooper

2019

Anal. Chem.

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Native ambient mass spectrometry has the potential for simultaneous analysis of native protein structure and spatial distribution within thin tissue sections. Notwithstanding sensitivity, this information can, in principle, be obtained for any protein present with no requirement for a priori knowledge of protein identity. To date, native ambient mass spectrometry has primarily made use of the liquid extraction surface analysis (LESA) sampling technique. Here, we address a fundamental question: Are the protein structures observed following native liquid extraction surface analysis representative of the protein structures within the substrate, or does the extraction process facilitate refolding (or unfolding)? Specifically, our aim was to determine whether protein–ligand complexes observed following LESA are indicative of complexes present in the substrate, or an artifact of the sampling process. The systems investigated were myoglobin and its noncovalently bound heme cofactor, and the Zn-binding protein carbonic anhydrase and its binding with ethoxzolamide. Charge state distributions, drift time profiles, and collision cross sections were determined by liquid extraction surface analysis ion mobility mass spectrometry of native and denatured proteins and compared with those obtained by direct infusion electrospray. The results show that it was not possible to refold denatured proteins with concomitant ligand binding (neither heme, zinc, nor ethoxzolamide) simply by use of native-like LESA solvents. That is, protein–ligand complexes were only observed by LESA MS when present in the substrate.

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“…CCS measurement of SOD1 ions. CCS values of SOD1 ions in the gas phase were measured and calculated according to a previously protocol 34 . Briefly, the CCS calibrants were subunits (β and γ) and trimer of the phospholipase A2 protein, Paradoxin (PDx), sourced from Taipan snake venom (Venom Supplies PTY LTD, Tanunda Australia) which were sprayed under similar conditions with the exception of the PDx (10 V Trap collision energy and 10 V Transfer collision energy).…”

Section: Sample Preparation For Mass Spectrometrymentioning

confidence: 99%

Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt Bridge Formation

et al. 2019

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Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel are explored by activation of protein ions in vacuum. Here we examine the trajectory that dimeric Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in vacuum. We examined wild-type SOD1 and six disease-related point-mutants by using tandem MS and ion-mobility MS (MS/MS-IMMS) coupled with increasing collisional activation potentials. For six of the seven SOD1 variants, increasing activation promoted dimers to transition through two unfolding events to access three gas-phase conformers before dissociating symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition, and displayed a much higher abundance of asymmetric products. We localise this effect to the formation of a new salt-bridge in the first activated conformation. To examine the data quantitatively, we generated a model of SOD1 gas phase unfolding and dissociation, and applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals an increase in the barrier height to unfolding in G37R to be >5 kJ/mol-1 higher than for the other variants, consistent with expectations for the strength of a salt-bridge. Our work demonstrates the importance of bond formation during the unfolding of proteins in vacuum, and provides a framework for comparing quantitatively the free energy landscape they explore upon activation. File list (2) download file view on ChemRxiv Main Text.pdf (1.37 MiB) download file view on ChemRxiv Supplementary Information.pdf (1.37 MiB)

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Conditions for Analysis of Native Protein Structures Using Uniform Field Drift Tube Ion Mobility Mass Spectrometry and Characterization of Stable Calibrants for TWIM-MS

Cited by 30 publications

References 45 publications

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies

Probing the Fundamentals of Native Liquid Extraction Surface Analysis Mass Spectrometry of Proteins: Can Proteins Refold during Extraction?

Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt Bridge Formation

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