Publications by authors named "David H Russell"

175 Publications

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding.

Anal Chem 2021 May 27;93(18):6924-6931. Epub 2021 Apr 27.

Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated. Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from ∼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold- and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.
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http://dx.doi.org/10.1021/acs.analchem.1c00870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119373PMC
May 2021

Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants.

Proc Natl Acad Sci U S A 2021 Mar;118(12)

Department of Chemistry, Texas A&M University, College Station, TX 77843;

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras-GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility-mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOS) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOS engaging KRas. We also find KRas exhibits high affinity for SOS and is a potent allosteric modulator of its activity. A structure of the KRas•SOS complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRas-GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas-GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRas•SOS complexes, underscoring the need for more potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.
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http://dx.doi.org/10.1073/pnas.2022403118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000204PMC
March 2021

Editorial: Focus on Ionization Technologies Used in MS: Fundamentals and Applications, Honoring Dr. Sarah Trimpin, Recipient of the 2019 ASMS Biemann Medal.

J Am Soc Mass Spectrom 2021 Mar;32(3):616-617

Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States.

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http://dx.doi.org/10.1021/jasms.1c00030DOI Listing
March 2021

Development of Native MS Capabilities on an Extended Mass Range Q-TOF MS.

Int J Mass Spectrom 2020 Dec 9;458. Epub 2020 Oct 9.

Department of Chemistry, Texas A&M University, College Station, TX 77843.

Native mass spectrometry (nMS) is increasingly used for studies of large biomolecules (>100 kDa), especially proteins and protein complexes. The growth in this area can be attributed to advances in native electrospray ionization as well as instrumentation that is capable of accessing high mass-to-charge () regimes without significant losses in sensitivity and resolution. Here, we describe modifications to the ESI source of an Agilent 6545XT Q-TOF MS that is tailored for analysis of large biomolecules. The modified ESI source was evaluated using both soluble and membrane protein complexes ranging from ~127 to ~232 kDa and the ~801 kDa protein chaperone GroEL. The increased mass resolution of the instrument affords the ability to resolve small molecule adducts and analyze collision-induced dissociation products of the native complexes.
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http://dx.doi.org/10.1016/j.ijms.2020.116451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641504PMC
December 2020

Evidence for Many Unique Solution Structures for Chymotrypsin Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS Measurements.

J Am Chem Soc 2020 10 29;142(41):17372-17383. Epub 2020 Sep 29.

Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, Indiana 47401, United States.

Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of Δ as well as Δ, Δ, and Δ for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.
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http://dx.doi.org/10.1021/jacs.0c05365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702223PMC
October 2020

Discovery of Potent Charge-Reducing Molecules for Native Ion Mobility Mass Spectrometry Studies.

Anal Chem 2020 08 31;92(16):11242-11249. Epub 2020 Jul 31.

Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

There is growing interest in the characterization of protein complexes and their interactions with ligands using native ion mobility mass spectrometry. A particular challenge, especially for membrane proteins, is preserving noncovalent interactions and maintaining native-like structures. Different approaches have been developed to minimize activation of protein complexes by manipulating charge on protein complexes in solution and the gas-phase. Here, we report the utility of polyamines that have exceptionally high charge-reducing potencies with some molecules requiring 5-fold less than trimethylamine oxide to elicit the same effect. The charge-reducing molecules do not adduct to membrane protein complexes and are also compatible with ion-mobility mass spectrometry, paving the way for improved methods of charge reduction.
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http://dx.doi.org/10.1021/acs.analchem.0c01826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056390PMC
August 2020

First-Principles Collision Cross Section Measurements of Large Proteins and Protein Complexes.

Anal Chem 2020 08 28;92(16):11155-11163. Epub 2020 Jul 28.

Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

Rotationally averaged collision cross section (CCS) values for a series of proteins and protein complexes ranging in size from 8.6 to 810 kDa are reported. The CCSs were obtained using a native electrospray ionization drift tube ion mobility-Orbitrap mass spectrometer specifically designed to enhance sensitivity while having high-resolution ion mobility and mass capabilities. Periodic focusing (PF)-drift tube (DT)-ion mobility (IM) provides first-principles determination of the CCS of large biomolecules that can then be used as CCS calibrants. The experimental, first-principles CCS values are compared to previously reported experimentally determined and computationally calculated CCS using projected superposition approximation (PSA), the Ion Mobility Projection Approximation Calculation Tool (IMPACT), and Collidoscope. Experimental CCS values are generally in agreement with previously reported CCSs, with values falling within ∼5.5%. In addition, an ion mobility resolution (CCS centroid divided by CCS fwhm) of ∼60 is obtained for pyruvate kinase (MW ∼ 233 kDa); however, ion mobility resolution for bovine serum albumin (MW ∼ 68 kDa) is less than ∼20, which arises from sample impurities and underscores the importance of sample quality. The high resolution afforded by the ion mobility-Orbitrap mass analyzer provides new opportunities to understand the intricate details of protein complexes such as the impact of post-translational modifications (PTMs), stoichiometry, and conformational changes induced by ligand binding.
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http://dx.doi.org/10.1021/acs.analchem.0c01285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7967297PMC
August 2020

THE IMS PARADOX: A PERSPECTIVE ON STRUCTURAL ION MOBILITY-MASS SPECTROMETRY.

Mass Spectrom Rev 2021 05 1;40(3):280-305. Epub 2020 Jul 1.

Department of Chemistry, Texas A&M University, College Station, TX, 77843.

Studies of large proteins, protein complexes, and membrane protein complexes pose new challenges, most notably the need for increased ion mobility (IM) and mass spectrometry (MS) resolution. This review covers evolutionary developments in IM-MS in the authors' and key collaborators' laboratories with specific focus on developments that enhance the utility of IM-MS for structural analysis. IM-MS measurements are performed on gas phase ions, thus "structural IM-MS" appears paradoxical-do gas phase ions retain their solution phase structure? There is growing evidence to support the notion that solution phase structure(s) can be retained by the gas phase ions. It should not go unnoticed that we use "structures" in this statement because an important feature of IM-MS is the ability to deal with conformationally heterogeneous systems, thus providing a direct measure of conformational entropy. The extension of this work to large proteins and protein complexes has motivated our development of Fourier-transform IM-MS instruments, a strategy first described by Hill and coworkers in 1985 (Anal Chem, 1985, 57, pp. 402-406) that has proved to be a game-changer in our quest to merge drift tube (DT) and ion mobility and the high mass resolution orbitrap MS instruments. DT-IMS is the only method that allows first-principles determinations of rotationally averaged collision cross sections (CSS), which is essential for studies of biomolecules where the conformational diversities of the molecule precludes the use of CCS calibration approaches. The Fourier transform-IM-orbitrap instrument described here also incorporates the full suite of native MS/IM-MS capabilities that are currently employed in the most advanced native MS/IM-MS instruments. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.
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http://dx.doi.org/10.1002/mas.21642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989064PMC
May 2021

Ag Ion Binding to Human Metallothionein-2A Is Cooperative and Domain Specific.

Anal Chem 2020 07 24;92(13):8923-8932. Epub 2020 Jun 24.

Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biological roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chemical labeling using -ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag-MT. The results for Ag-MT are then compared to similar results obtained for Cd-MT. The results show that Ag-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry analysis combined with NEM labeling revealed that all four Ag ions of Ag-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag and Cd react with MT to yield cooperative products, i.e., Ag-MT and Cd-MT, these products are very different; Ag ions of Ag-MT are located in the β-domain, whereas Cd ions of Cd-MT are located in the α-domain. Ag-MT has been reported to be fully metalated in the β-domain, but our data suggest the two additional Ag ions are more weakly bound than are the other four. Higher order Ag-MT complexes ( = 7-17) are formed in solutions that contain excess Ag ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag ions are displaced upon addition of NEM to this solution to yield predominantly AgNEM-MT. Results from CIU suggest that Ag-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag increases.
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http://dx.doi.org/10.1021/acs.analchem.0c00829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114364PMC
July 2020

Collision-Induced Unfolding Studies of Proteins and Protein Complexes using Drift Tube Ion Mobility-Mass Spectrometer.

Anal Chem 2020 05 8;92(10):7218-7225. Epub 2020 May 8.

Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

Elucidating the structures and stabilities of proteins and their complexes is paramount to understanding their biological functions in cellular processes. Native mass spectrometry (MS) coupled with ion mobility spectrometry (IMS) is emerging as an important biophysical technique owing to its high sensitivity, rapid analysis time, and ability to interrogate sample complexity or heterogeneity and the ability to probe protein structure dynamics. Here, a commercial IMS-MS platform has been modified for static native ESI emitters and an extended mass-to-charge range (20 kDa /) and its performance capabilities and limits were explored for a range of protein and protein complexes. The results show new potential for this instrument platform for studies of large protein and protein complexes and provides a roadmap for extending the performance metrics for studies of even larger, more complex systems, namely, membrane protein complexes and their interactions with ligands.
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http://dx.doi.org/10.1021/acs.analchem.0c00772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289629PMC
May 2020

Native IM-Orbitrap MS: Resolving What Was Hidden.

Trends Analyt Chem 2020 Mar 31;124. Epub 2019 May 31.

Department of Chemistry, Texas A&M University, 3255 TAMU, College Station, Texas 77843.

Native ion mobility-mass spectrometry (IM-MS) is an emerging biophysical approach to probe the intricate details of protein structure and function. The instrument design enables measurements of accurate first-principle determinations of rotationally-averaged ion-neutral collision cross sections coupled with high-mass, high-resolution mass measurement capabilities of Orbitrap MS. The inherent duty-cycle mismatch between drift tube IM and Orbitrap MS is alleviated by operating the drift tube in a frequency modulated mode while continuously acquiring mass spectra with the Orbitrap MS. Fourier transform of the resulting time-domain signal, i.e., ion abundances as a function of the modulation frequency, yields a frequency domain spectrum that is then converted (s to s) to IM drift time. This multiplexed approach allows for a duty-cycle of 25% compared to <1% for traditional "pulse-and-wait" IM-ToF-MS. Improvements in mobility and mass resolution of the IM-Orbitrap allows for accurate analysis of intact protein complexes and the possibility of capturing protein dynamics.
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http://dx.doi.org/10.1016/j.trac.2019.05.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079669PMC
March 2020

Molecular Mechanism of ISC Iron-Sulfur Cluster Biogenesis Revealed by High-Resolution Native Mass Spectrometry.

J Am Chem Soc 2020 04 17;142(13):6018-6029. Epub 2020 Mar 17.

Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States.

Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.
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http://dx.doi.org/10.1021/jacs.9b11454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262672PMC
April 2020

Structural Analysis of the Effect of a Dual-FLAG Tag on Transthyretin.

Biochemistry 2020 03 2;59(9):1013-1022. Epub 2020 Mar 2.

Department of Chemistry, Texas A & M University, College Station, Texas 77843, United States.

Recombinant proteins have increased our knowledge regarding the physiological role of proteins; however, affinity purification tags are often not cleaved prior to analysis, and their effects on protein structure, stability and assembly are often overlooked. In this study, the stabilizing effects of an N-terminus dual-FLAG (FT) tag fusion to transthyretin (TTR), a construct used in previous studies, are investigated using native ion mobility-mass spectrometry (IM-MS). A combination of collision-induced unfolding and variable-temperature electrospray ionization is used to compare gas- and solution-phase stabilities of FT-TTR to wild-type and C-terminal tagged TTR. Despite an increased stability of both gas- and solution-phase FT-TTR, thermal degradation of FT-TTR was observed at elevated temperatures, viz., backbone cleavage occurring between Lys9 and Cys10. This cleavage reaction is consistent with previously reported metalloprotease activity of TTR [Liz et al. 2009] and is suppressed by either metal chelation or excess zinc. This study brings to the fore the effect of affinity tag stabilization of TTR and emphasizes unprecedented detail afforded by native IM-MS to assess structural discrepancies of recombinant proteins from their wild-type counterparts.
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http://dx.doi.org/10.1021/acs.biochem.0c00105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171973PMC
March 2020

Tracking the Structural Evolution of 4-Aminobenzoic Acid in the Transition from Solution to the Gas Phase.

J Phys Chem B 2020 03 6;124(11):2081-2087. Epub 2020 Mar 6.

Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.

Here, cryogenic ion mobility-mass spectrometry (cryo-IM-MS) is used to investigate intracluster proton transfer reactions of 4-aminobenzoic acid during the transition from solution to the gas phase. Previous studies have shown that protonation of the amine group of 4-aminobenzoic acid (4-ABAH) is favored in solution (N-protomer), whereas protonation of the carboxylic acid group is favored in the gas phase (O-protomer). Results from cryo-IM-MS (80 K) studies of hydrated 4-ABAH ions, 4-ABAH(HO), are interpreted as evidence that the proton transfer reaction occurs through a water bridge at = 6 connecting the -NH and -COOH groups, that is a Grotthuss mechanism. The weak binding energy of water molecules imposes limits for obtaining first-principles collisional cross sections (CCSs) of hydrated ions; consequently, candidate structures for 4-ABAH(HO) ions are derived by correlating experimental arrival-time distributions to theoretically determined CCSs. To our knowledge, these are the first first-principles determinations of CCS for hydrated ions. Apolar cosolvents, particularly acetonitrile, have been postulated to inhibit proton transfer by blocking the Grotthuss mechanism, but our data suggest that acetonitrile simply stabilizes the ammonium ion.
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http://dx.doi.org/10.1021/acs.jpcb.9b10576DOI Listing
March 2020

Melting of Hemoglobin in Native Solutions as measured by IMS-MS.

Anal Chem 2020 02 7;92(4):3440-3446. Epub 2020 Feb 7.

Department of Chemistry , Indiana University , Bloomington , Indiana 47405 , United States.

Thermally induced structural transitions of the quaternary structure of the hemoglobin tetramer (human) in aqueous solution (150 mM ammonium acetate) were investigated using a variable temperature electrospray ionization (vt-ESI) technique in combination with ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements. At low solution temperatures (28 to ∼40 °C), a heterotetrameric (αβ) complex is the most abundant species that is observed. When the solution temperature is increased, this assembly dissociates into heterodimers (holo αβ forms) before ultimately forming insoluble aggregates at higher temperatures (>60 °C). In addition to the holo αβ forms, a small population of αβ dimers containing only a single heme ligand and having a dioxidation modification mapping to the β subunit are observed. The oxidized heterodimers are less stable than the unmodified holo-heterodimer. The Cys residue of the β subunit is the primary site of dioxidation. The close proximity of this post translational modification to both the αβ subunit interface and the heme binding site suggests that this modification is coupled to the loss of the heme and decreased protein stability. Changes in the charge state and collision cross sections of these species indicate that the tetramers and dimers favor less compact structures at elevated temperatures (prior to temperatures where dissociation dominates).
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http://dx.doi.org/10.1021/acs.analchem.9b05561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480357PMC
February 2020

Structural Analysis of 14-3-3-ζ-Derived Phosphopeptides Using Electron Capture Dissociation Mass Spectrometry, Traveling Wave Ion Mobility Spectrometry, and Molecular Modeling.

J Phys Chem B 2020 01 9;124(3):461-469. Epub 2020 Jan 9.

Centre of Membrane Proteins and Receptors (COMPARE) , Universities of Birmingham and Nottingham , Midlands , U.K.

Previously, we have demonstrated the effect of salt bridges on the electron capture dissociation mass spectrometry behavior of synthetic model phosphopeptides and applied an ion mobility spectrometry/molecular modeling approach to rationalize the findings in terms of peptide ion structure. Here, we develop and apply the approach to a biologically derived phosphopeptide. Specifically, we have investigated variants of a 15-mer phosphopeptide VVGARRSsWRVVSSI (s denotes phosphorylated Ser) derived from Akt1 substrate 14-3-3-ζ, which contains the phosphorylation motif RRSsWR. Variants were generated by successive arginine-to-leucine substitutions within the phosphorylation motif. ECD fragmentation patterns for the eight phosphopeptide variants show greater sequence coverage with successive R → L substitutions. Peptides with two or more basic residues had regions with no sequence coverage, while full sequence coverage was observed for peptides with one or no basic residues. For three of the peptide variants, low-abundance fragments were observed between the phosphoserine and a basic residue, possibly due to the presence of multiple conformers with and without noncovalent interactions between these residues. For the five variants whose dissociation behavior suggested the presence of intramolecular noncovalent interactions, we employed ion mobility spectrometry and molecular modeling to probe the nature of these interactions. Our workflow allowed us to propose candidate structures whose noncovalent interactions were consistent with the ECD data for all of the peptides modeled. Additionally, the AMBER parameter sets created for and validated by this work are presented and made available online ( http://www.biosciences-labs.bham.ac.uk/cooper/datasets.php ).
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http://dx.doi.org/10.1021/acs.jpcb.9b08506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241667PMC
January 2020

Topological Characterization of Coordination-Driven Self-assembly Complexes: Applications of Ion Mobility-Mass Spectrometry.

J Am Soc Mass Spectrom 2019 Sep 17;30(9):1654-1662. Epub 2019 Jul 17.

Department of Chemistry, Texas A&M University, College Station, TX, 77843, USA.

Coordination-driven self-assembly (CDSA) is increasingly used to synthesize coordination complexes containing metal-centered electron acceptors and typically nitrogen-containing electron donors. Characterization of the structures obtained from CDSA via crystallographic or spectroscopic means is limited due to difficulties in forming single crystals for X-ray studies and overlapping precursor and product signals in NMR. Here, we employ ion mobility-mass spectrometry (IM-MS), which provides a direct measure of size and shape of the CDSA complexes, to study the intact reaction products of a rhomboid-shaped complex. This approach negates the need for product isolation and crystallization and allows for tracking of the product distribution as a function of time. A potential challenge of IM-MS is that the size/shape of the observed CDSA complexes can vary with internal energy; however, we show that proper tuning of the instrument reduces the effects of collisional activation thereby allowing for retention of ion conformations that reflect solution-phase ion structures. Graphical Abstract.
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http://dx.doi.org/10.1007/s13361-019-02276-6DOI Listing
September 2019

Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry.

Biochemistry 2019 08 22;58(31):3396-3405. Epub 2019 Jul 22.

Department of Chemistry , Texas A&M University , College Station , Texas 77843 , United States.

Mutations in RAS are associated with many different cancers and have been a therapeutic target for more than three decades. RAS cycles from an active to inactive state by both intrinsic and GTPase-activating protein (GAP)-stimulated hydrolysis. The activated enzyme interacts with downstream effectors, leading to tumor proliferation. Mutations in RAS associated with cancer are insensitive to GAP, and the rate of inactivation is limited to their intrinsic hydrolysis rate. Here, we use high-resolution native mass spectrometry (MS) to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for K-RAS and its oncogenic mutants. MS data reveal heterogeneity where both 2'-deoxy and 2'-hydroxy forms of GDP (guanosine diphosphate) and GTP (guanosine triphosphate) are bound to the recombinant enzyme. Intrinsic GTPase activity is directly monitored by the loss in mass of K-RAS bound to GTP, which corresponds to the release of phosphate. The rates determined from MS are in direct agreement with those measured using an established solution-based assay. Our results show that the transition state thermodynamics for the intrinsic GTPase activity of K-RAS is both enthalpically and entropically unfavorable. The oncogenic mutants G12C, Q61H, and G13D unexpectedly exhibit a 2'-deoxy GTP intrinsic hydrolysis rate higher than that for GTP.
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http://dx.doi.org/10.1021/acs.biochem.9b00532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196336PMC
August 2019

Variable-Temperature ESI-IMS-MS Analysis of Myohemerythrin Reveals Ligand Losses, Unfolding, and a Non-Native Disulfide Bond.

Anal Chem 2019 05 9;91(10):6808-6814. Epub 2019 May 9.

Department of Chemistry , Indiana University , Bloomington , Indiana 47405 , United States.

Variable-temperature electrospray ionization combined with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques are used to monitor structural transitions of the protein myohemerythrin from peanut worm in aqueous ammonium acetate solutions from ∼15 to 92 °C. At physiological temperatures, myohemerythrin favors a four-helix bundle motif and has a diiron oxo cofactor that binds oxygen. As the solution temperature is increased from ∼15 to 35 °C, some bound oxygen dissociates; at ∼66 °C, the cofactor dissociates to produce populations of both folded and unfolded apoprotein. At higher temperatures (∼85 °C and above), the IMS-MS spectrum indicates that the folded apoprotein dominates, and provides evidence for stabilization of the structure by formation of a non-native disulfide bond. In total, we find evidence for 18 unique forms of myohemerythrin as well as information about the structures and stabilities of these states. The high-fidelity of IMS-MS techniques provides a means of examining the stabilities of individual components of complex mixtures that are inaccessible by traditional calorimetric and spectroscopic methods.
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http://dx.doi.org/10.1021/acs.analchem.9b00981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935875PMC
May 2019

Substance P in Solution: Trans-to-Cis Configurational Changes of Penultimate Prolines Initiate Non-enzymatic Peptide Bond Cleavages.

J Am Soc Mass Spectrom 2019 Jun 12;30(6):919-931. Epub 2019 Apr 12.

Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, IN, 47401, USA.

We report ion mobility spectrometry and mass spectrometry studies of the non-enzymatic step-by-step degradation of substance P (subP), an 11-residue neuropeptide, with the sequence Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH, in ethanol. At elevated solution temperatures (55 to 75 °C), several reactions are observed, including a protonation event, i.e., [subP+2H] + H → [subP+3H], that appears to be regulated by a configurational change and two sequential bond cleavages (the Pro-Lys peptide bond is cleaved to form the smaller nonapeptide Lys-Met-NH [subP], and subsequently, subP is cleaved at the Pro-Gln peptide bond to yield the heptapeptide Gln-Met-NH [subP]). Each of the product peptides [subP and subP] is accompanied by a complementary diketopiperazine (DKP): cyclo-Arg-Pro (cRP) for the first cleavage, and cyclo-Lys-Pro (cKP) for the second. Insight about the mechanism of degradation is obtained by comparing kinetics calculations of trial model mechanisms with experimental data. The best model of our experimental data indicates that the initial cleavage of subP is regulated by a conformational change, likely a trans→cis isomerization of the Arg-Pro peptide bond. The subP product has a long lifetime (t ~ 30 h at 55 °C) and appears to transition through several structural intermediates prior to dissociation, suggesting that subP is initially formed with a Lys-trans-Pro peptide bond configuration and that slow trans→cis isomerization regulates the second bond cleavage event as well. From these data and our model mechanisms, we obtain transition state thermochemistry ranging from ΔH = 41 to 85 kJ mol and ΔS = - 43 to - 157 J mol K for each step in the reaction. Graphical Abstract.
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http://dx.doi.org/10.1007/s13361-019-02159-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824264PMC
June 2019

Substance P in the Gas Phase: Conformational Changes and Dissociations Induced by Collisional Activation in a Drift Tube.

J Am Soc Mass Spectrom 2019 Jun 12;30(6):932-945. Epub 2019 Apr 12.

Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, IN, 47401, USA.

The work presented below is related to our companion paper in this issue, entitled: Substance P in solution: trans-to-cis configurational changes of penultimate prolines initiate non-enzymatic peptide bond cleavages. Two-dimensional ion mobility spectrometry (IMS-IMS) and mass spectrometry techniques are used to investigate structural transitions for [M+3H] ions of substance P (subP) upon collisional activation (CA) in the gas phase. In this approach, different conformations of ions having a specified mobility are selected after an initial IMS separation, collisionally activated to produce new conformers, and these product structures are separated again using a second IMS region. In this way, it is possible to follow folding and unfolding transitions of different conformations. The analysis shows evidence for five conformations. Unlike other systems, every transition is irreversible. Studies as a function of activation voltage are used to discern pathways of structural changes prior to reaching the energy required for dissociation. Thresholds associated with the onsets of transitions are calibrated to obtain estimates of the energetic barriers between different structures and semi-quantitative potential energy diagrams are presented. Overall, barriers associated with structural transitions of [subP+3H] in the absence of solvent are on the order of ~ 40 kJ mol, substantially lower than the ~ 90 kJ mol required for some similar structural transitions in solutions of ethanol. Comparisons of the transition energies in the gas phase with thermochemistry for similar transitions in solution provide clues about why reverse transitions are prohibited. Graphical Abstract.
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http://dx.doi.org/10.1007/s13361-019-02160-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6865269PMC
June 2019

Selective binding of a toxin and phosphatidylinositides to a mammalian potassium channel.

Nat Commun 2019 03 22;10(1):1352. Epub 2019 Mar 22.

Department of Chemistry, Texas A&M University, College Station, TX, 77842, USA.

G-protein-gated inward rectifying potassium channels (GIRKs) require G subunits and phosphorylated phosphatidylinositides (PIPs) for gating. Although studies have provided insight into these interactions, the mechanism of how these events are modulated by G and the binding affinity between PIPs and GIRKs remains poorly understood. Here, native ion mobility mass spectrometry is employed to directly monitor small molecule binding events to mouse GIRK2. GIRK2 binds the toxin tertiapin Q and PIPs selectively and with significantly higher affinity than other phospholipids. A mutation in GIRK2 that causes a rotation in the cytoplasmic domain, similarly to G-binding to the wild-type channel, revealed differences in the selectivity towards PIPs. More specifically, PIP isoforms known to weakly activate GIRKs have decreased binding affinity. Taken together, our results reveal selective small molecule binding and uncover a mechanism by which rotation of the cytoplasmic domain can modulate GIRK•PIP interactions.
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http://dx.doi.org/10.1038/s41467-019-09333-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430785PMC
March 2019

New insights into the metal-induced oxidative degradation pathways of transthyretin.

Chem Commun (Camb) 2019 Apr;55(28):4091-4094

Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA.

The amyloidogenic mechanism of transthyretin is still debated but understanding it fully could lend insight into disease progression and potential therapeutics. Transthyretin was investigated revealing a metal-induced (Cr/Cu) oxidation pathway leading to N-terminal backbone fragmentation and oligomer formation; previously hidden details were revealed only by FT-IM-Orbitrap MS and surface-induced dissociation.
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http://dx.doi.org/10.1039/c9cc00682fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452628PMC
April 2019

Hydration of Guanidinium Ions: An Experimental Search for Like-Charged Ion Pairs.

J Phys Chem Lett 2019 Mar 8;10(6):1349-1354. Epub 2019 Mar 8.

Department of Chemistry Texas A&M University College Station , Texas 77843 , United States.

Guanidinium ions (GdmH) are reported to form stable complexes (GdmH/GdmH) in aqueous solution despite strong repulsive interactions between the like-charged centers. These complexes are thought to play important roles in protein folding, membrane penetration, and formation of protein dimers. Although GdmH ions are weakly hydrated, semiempirical calculations provide evidence that these like-charged complexes are stabilized by water molecules, which serve important structural and energetic roles. Specifically, water molecules bridge between the GdmH ions of GdmH/GdmH complexes as well as complexes involving the guanidinium side chains of arginine. Potential biological significances of like-charged complexes have been largely confirmed by ab initio molecular dynamics simulations and indirect experimental evidence. We report cryo-ion mobility-mass spectrometry results for the GdmH/GdmH ion pair confined in a nanodroplet- the first direct experimental observation of this like-charged complex. A second like-charged complex, described as a water-mediated complex involving GdmH and HO, was also observed.
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http://dx.doi.org/10.1021/acs.jpclett.9b00268DOI Listing
March 2019

Topological Analysis of Transthyretin Disassembly Mechanism: Surface-Induced Dissociation Reveals Hidden Reaction Pathways.

Anal Chem 2019 02 28;91(3):2345-2351. Epub 2019 Jan 28.

Department of Chemistry , Texas A&M University , College Station , Texas 77843 , United States.

The proposed mechanism of fibril formation of transthyretin (TTR) involves self-assembly of partially unfolded monomers. However, the mechanism(s) of disassembly to monomer and potential intermediates involved in this process are not fully understood. In this study, native mass spectrometry and surface-induced dissociation (SID) are used to investigate the TTR disassembly mechanism(s) and the effects of temperature and ionic strength on the kinetics of TTR complex formation. Results from the SID of hybrid tetramers formed during subunit exchange provide strong evidence for a two-step mechanism whereby the tetramer dissociates to dimers that then dissociate to monomers. Also, the SID results uncovered a hidden pathway in which a specific topology of the hybrid tetramer is directly produced by assembly of dimers in the early steps of TTR disassembly. Implementation of SID to dissect protein topology during subunit exchange provides unique opportunities to gain unparalleled insight into disassembly pathways.
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http://dx.doi.org/10.1021/acs.analchem.8b05066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6464633PMC
February 2019

A Focus Honoring Carol Robinson's Election to the National Academy of Sciences.

J Am Soc Mass Spectrom 2019 Jan;30(1):1-3

Department of Chemistry, Texas A&M University, College Station, TX, USA.

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http://dx.doi.org/10.1007/s13361-018-2096-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7097593PMC
January 2019

Collision-Induced Unfolding of Partially Metalated Metallothionein-2A: Tracking Unfolding Reactions of Gas-Phase Ions.

Anal Chem 2018 10 1;90(20):11856-11862. Epub 2018 Oct 1.

Department of Chemistry , Texas A&M University , College Station , Texas 77843 , United States.

Metallothioneins (MTs) constitute a group of intrinsically disordered proteins that exhibit extreme diversity in structure, biological functionality, and metal ion specificity. Structures of coordinatively saturated metalated MTs have been extensively studied, but very limited structural information for the partially metalated MTs exists. Here, the conformational preferences from partial metalation of rabbit metallothionein-2A (MT) by Cd, Zn, and Ag are studied using nanoelectrospray ionization ion mobility mass spectrometry. We also employ collision-induced unfolding to probe differences in the gas-phase stabilities of these partially metalated MTs. Our results show that despite their similar ion mobility profiles, Cd-MT, Zn-MT, Ag-MT, and Ag-MT differ dramatically in their gas-phase stabilities. Furthermore, the sequential addition of each Cd and Zn ion results in the incremental stabilization of unique unfolding intermediates.
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http://dx.doi.org/10.1021/acs.analchem.8b01622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463490PMC
October 2018

Fourier Transform-Ion Mobility-Orbitrap Mass Spectrometer: A Next-Generation Instrument for Native Mass Spectrometry.

Anal Chem 2018 09 22;90(17):10472-10478. Epub 2018 Aug 22.

Department of Chemistry , Texas A&M University , College Station , Texas 77843 , United States.

A new instrument configuration for native ion mobility-mass spectrometry (IM-MS) is described. Macromolecule ions are generated by using a static ESI source coupled to an RF ion funnel, and these ions are then mobility and mass analyzed using a periodic focusing drift tube IM analyzer and an Orbitrap mass spectrometer. The instrument design retains the capabilities for first-principles determination of rotationally averaged ion-neutral collision cross sections and high-resolution measurements in both mobility and mass analysis modes for intact protein complexes. Operation in the IM mode utilizes FT-IMS modes (originally described by Knorr ( Knorr , F. J. Anal. Chem . 1985 , 57 ( 2 ), 402 - 406 )), which provides a means to overcome the inherent duty cycle mismatch for drift tube (DT)-IM and Orbitrap mass analysis. The performance of the native ESI-FT-DT-IM-Orbitrap MS instrument was evaluated using the protein complexes Gln K (MW 44 kDa) and streptavidin (MW 53 kDa) bound to small molecules (ADP and biotin, respectively) and transthyretin (MW 56 kDa) bound to thyroxine and zinc.
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http://dx.doi.org/10.1021/acs.analchem.8b02463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6464636PMC
September 2018

Solvent Mediation of Peptide Conformations: Polyproline Structures in Water, Methanol, Ethanol, and 1-Propanol as Determined by Ion Mobility Spectrometry-Mass Spectrometry.

J Am Soc Mass Spectrom 2019 Jan 1;30(1):77-84. Epub 2018 Aug 1.

Department of Chemistry, Indiana University, 800 Kirkwood Avenue, Bloomington, IN, 47401, USA.

Ion mobility spectrometry and circular dichroism spectroscopy are used to examine the populations of the small model peptide, polyproline-13 in water, methanol, ethanol, and 1-propanol over a range of solution temperatures (from 288 to 318 K). At low temperatures, the less-polar solvents (1-propanol and ethanol) favor the all-cis polyproline I helix (PPI); as the temperature is increased, the trans-configured polyproline II helix (PPII) is formed. In polar solvents (methanol and water), PPII is favored at all temperatures. From the experimental data, we determine the relative stabilities of the eight structures in methanol, ethanol, and 1-propanol, as well as four in water, all with respect to PPII. Although these conformers show relatively small differences in free energies, substantial variability is observed in the enthalpies and entropies across the structures and solvents. This requires that enthalpies and entropies be highly correlated: in 1-propanol, cis-configured PPI conformations are energetically favorable but entropically disfavored. In more polar solvents, PPI is enthalpically less favorable and entropy favors trans-configured forms. While either ΔH or ΔS can favor different structures, no conformation in any solvent is simultaneously energetically and entropically stabilized. These data present a rare opportunity to examine the origin of conformational stability. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-2034-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503664PMC
January 2019