Melting Proteins: Evidence for Multiple Stable Structures upon Thermal Denaturation of Native Ubiquitin from Ion Mobility Spectrometry-Mass Spectrometry Measurements

El‐Baba, Tarick J.; Woodall, Daniel W.; Raab, Shannon A.; Fuller, Daniel R.; Laganowsky, Arthur; Russell, David H.; Clemmer, David E.

doi:10.1021/jacs.7b02774

Cited by 109 publications

(170 citation statements)

References 44 publications

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“…The Clemmer group reported the retention of native compact conformers for the same dominant charge states observed in our experiment [38, 39]. In the current configuration and solution conditions, the 3 + charge state was observed and is lower than reported by our group and others, suggesting the REIS is a “cold” source [6, 40, 41].…”

Section: Resultssupporting

confidence: 80%

Development and Evaluation of a Reverse-Entry Ion Source Orbitrap Mass Spectrometer

Poltash

McCabe

Patrick

et al. 2018

J. Am. Soc. Mass Spectrom.

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As a step towards development of a high-resolution ion mobility mass spectrometer using the orbitrap mass analyzer platform, we describe herein a novel reverse-entry ion source (REIS) coupled to the higher-energy C-trap dissociation (HCD) cell of an orbitrap mass spectrometer with extended mass range. Development of the REIS is a first step in the development of a drift tube ion mobility-orbitrap MS. The REIS approach retains the functionality of the commercial instrument ion source which permits the uninterrupted use of the instrument during development as well as performance comparisons between the two ion sources. Ubiquitin (8.5 kDa) and lipid binding to the ammonia transport channel (AmtB, 126 kDa) protein complex were used as model soluble and membrane proteins, respectively, to evaluate the performance of the REIS instrument. Mass resolution obtained with the REIS is comparable to that obtained using the commercial ion source. The charge state distributions for ubiquitin and AmtB obtained on the REIS are in agreement with previous studies which suggests that the REIS-orbitrap EMR retains native structure in the gas phase. Graphical Abstract ᅟ.

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Section: Resultssupporting

confidence: 80%

Development and Evaluation of a Reverse-Entry Ion Source Orbitrap Mass Spectrometer

Poltash

McCabe

Patrick

et al. 2018

J. Am. Soc. Mass Spectrom.

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“…To do so, we built a temperature-variable nanoelectrospray source, inspired by previous designs. [40][41][42][43] The nanospray emitter is embedded inside a temperature-controlled copper block ( Figure S 1). MS-melting experiments,…”

Section: Abbreviationmentioning

confidence: 99%

Thermal Denaturation of DNA G-Quadruplexes and their Complexes with Ligands: Thermodynamic Analysis of the Multiple States Revealed by Mass Spectrometry

Marchand

Rosu

Zenobi

et al. 2018

Preprint

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As the idea that G-quadruplex nucleic acid structures are involved in cellular processes is gaining support, it becomes important to develop ligands that specifically target G-quadruplexes. However, ligand design is complicated because there are multiple G-quadruplex target sequences, some sequences are polymorphic, and very few ligand-quadruplex structures in solution were solved to date. Further, structure alone does not reveal the driving forces for ligand binding. To know why a ligand binds, the thermodynamics of binding must be characterized. Electrospray mass spectrometry makes it possible to detect and quantify each specific stoichiometry in terms of number of strands, number of specific cations, and number of ligands, and thus allows one to simultaneously determine the equilibrium constants for the formation of each complex. We designed and built a temperature-controlled nano-electrospray source to monitor thermal denaturation by mass spectrometry ("MS-melting"). We studied the thermal denaturation of G-quadruplexes, including the c-myc promoter and several telomeric sequence variants, and their complexes with popular ligands (Phen-DC3, TrisQ, TMPyP4, Cu-ttpy). From the temperature dependence of the equilibrium constants, we determined the enthalpic and entropic contributions to the formation of each stoichiometric state. In absence of ligand, we untangled the potassium-induced G-quadruplex folding thermodynamics, one potassium ion at a time. The formation of each quartet-K + -quartet units is strongly enthalpy driven, with entropy penalty. In contrast, the formation of quartet-K + -triplet units is entropically driven. For this reason, such misfolded structures can become more abundant as the temperature increases.In the presence of ligands, mass spectrometry also revealed new states at intermediate temperatures. For example, even in cases where only a 1:1 (ligand:quadruplex) is observed at room temperature, a 2:1 complex predominates at intermediate temperatures. Mass spectrometry also makes it easy to distinguish ligand bound to the 2-quartet structures (containing 1 K + ), the 3-quartet structures (containing 2 K + ) and to the unfolded strand (no specific K + ). We confirm that TrisQ binds preferably, but not exclusively, to 3quartet structures, Phen-DC3 binds to a 2-quartet structure, while the porphyrin ligand TMPyP4 is characterized as non-selective, because it binds to all forms including the unfolded one. The thermodynamics of ligand binding to each form, one ligand at a time, provides unprecedented detail on the interplay between ligand binding and changes in G-quadruplex topology.

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“…As electrospray generally preserves intramolecular non-covalent interactions, and as ion mobility spectrometry probes gas-phase compactness, it seems logical that ion mobility spectrometry should probe the compactness of solution phase structures. Several studies have validated this logic by showing that folded structures in solution end up compact in the gas phase, while unfolded structures in solution end up much more extended in the gas phase [21][22][23].…”

Section: Introductionmentioning

confidence: 91%

Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process

Khristenko

Amato

Livet

et al. 2019

J. Am. Soc. Mass Spectrom.

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Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding.Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C-H + -C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue-type pathway or a chain extrusion-type pathway.

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Melting Proteins: Evidence for Multiple Stable Structures upon Thermal Denaturation of Native Ubiquitin from Ion Mobility Spectrometry-Mass Spectrometry Measurements

Cited by 109 publications

References 44 publications

Development and Evaluation of a Reverse-Entry Ion Source Orbitrap Mass Spectrometer

Development and Evaluation of a Reverse-Entry Ion Source Orbitrap Mass Spectrometer

Thermal Denaturation of DNA G-Quadruplexes and their Complexes with Ligands: Thermodynamic Analysis of the Multiple States Revealed by Mass Spectrometry

Native Ion Mobility Mass Spectrometry: When Gas-Phase Ion Structures Depend on the Electrospray Charging Process

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