The effect of salt on the conformations of three model proteins is revealed by variable temperature ion mobility mass spectrometry

Berezovskaya, Yana; Porrini, Massimiliano; Barran, Perdita E.

doi:10.1016/j.ijms.2013.02.005

Cited by 26 publications

(48 citation statements)

References 55 publications

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“…By lowering the drift cell temperature to 200 K we are able to 'freeze out' conformations which would not otherwise be observable. At this temperature, we report a wider CCS distribution for all charge states and an increased CCS for z < 8, a phenomenon discussed elsewhere 23,25 . Compared with the trends reported for cytochrome c and p53 DBD, for MDM2 we observe charge state dependent unfolding pathways.…”

Section: N-terminal Mdm2 Discussionsupporting

confidence: 79%

“…[19][20][21][22] This approach was taken by both Jarrold, and Bowers, who used collisional activation provided by increasing injection energies 21 or temperature 11,12,23,24 to probe the unfolding transitions of various proteins by IM-MS. More recently, protein unfolding was modulated by addition of salt molecules and investigated by IM-MS as a function of temperature. 25 Whilst most studies regarding the effect of temperature on protein structure are performed in the solution phase, where the protein is stabilised by both intramolecular interactions and interactions between the protein and its surrounding solvent, gas phase techniques utilise solvent free charged analyte ions, thus removing all hydration effects and allowing intramolecular interactions upon unfolding to be probed. For some proteins the effect of temperature on structure is difficult to study in a solution phase assay since thermally induced structural transitions are coupled to aggregation.…”

Section: Introductionmentioning

confidence: 99%

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Insights into the Conformations of Three Structurally Diverse Proteins: Cytochrome c, p53, and MDM2, Provided by Variable-Temperature Ion Mobility Mass Spectrometry

et al. 2015

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Thermally induced conformational transitions of three proteins of increasing intrinsic disorder-cytochrome c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)-have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome c, dimers of significant intensity are also observed. Cytochrome c [M + 5H](5+) ions present in one conformer of CCS ∼1200 Å(2), undergoing compaction in line with the reported Tmelt = 360.15 K before slight unfolding at 571 K. The more extended [M + 7H](7+) cytochrome c monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H](11+) dimer presents as two conformers, which undergo slight structural compaction or annealing before dissociation. p53 DBD follows a trend of structural collapse before an increase in the observed collision cross section (CCS), akin to that observed for cytochrome c but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS ∼1750 Å(2) at 365 K, in line with the low solution Tmelt = 315-317 K. The protein then extends to produce either a broad unresolved CCS distribution or, for z > 9, two conformers. NT-MDM2 exhibits a greater number of structural rearrangements, displaying charge-state-dependent unfolding pathways. DT-IM-MS experiments at 200 K resolve multiple conformers. Low charge state species of NT-MDM2 present as a single compact conformational family centered on CCS ∼1250 Å(2) at 300 K. This undergoes conformational tightening in line with the solution Tmelt = 348 K before unfolding at the highest temperatures. The more extended charge states present in two or more conformers at room temperature, undergoing thermally induced unfolding before significant structural collapse or annealing at high temperatures. Variable-temperature IM-MS is here shown to be an exciting approach to discern protein unfolding pathways for conformationally diverse proteins.

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Section: N-terminal Mdm2 Discussionsupporting

confidence: 79%

Section: Introductionmentioning

confidence: 99%

Insights into the Conformations of Three Structurally Diverse Proteins: Cytochrome c, p53, and MDM2, Provided by Variable-Temperature Ion Mobility Mass Spectrometry

et al. 2015

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“…[16,17] Variationsi ns tability between antibodies of the same subclass come from differences in the sequence of the variable domain. [22][23][24] VT-IM-MS measurementso nt hree intact antibodies with the same antigen binding specificity (IgG1, IgG4 and ah ybrid IgG4 with inserted IgG1 upper and core hinge, denoted IgG4DIgG1-hinge) as well as two Fc-hingef ragmentsa re reported here. [7,11] In this work, we exploit the hybrid technique of ion mobility mass spectrometry (IM-MS), which provides shape-defining parameters to terms of collisionc rosssections ( DT CCS He )a nd facilitates the study of the higher-order structure of proteins such as mAbs or their derivatives.…”

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confidence: 81%

Molecular Insights into the Thermal Stability of mAbs with Variable‐Temperature Ion‐Mobility Mass Spectrometry

et al. 2015

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The aggregation of protein-based therapeutics such as monoclonal antibodies (mAbs) can affect the efficacy of the treatment and can even induce effects that are adverse to the patient. Protein engineering is used to shift the mAb away from an aggregation-prone state by increasing the thermodynamic stability of the native fold, which might in turn alter conformational flexibility. We have probed the thermal stability of three types of intact IgG molecules and two Fc-hinge fragments by using variable-temperature ion-mobility mass spectrometry (VT-IM-MS). We observed changes in the conformations of isolated proteins as a function of temperature (300-550 K). The observed differences in thermal stability between IgG subclasses can be rationalized in terms of changes to higher-order structural organization mitigated by the hinge region. VT-IM-MS provides insights into mAbs structural thermodynamics and is presented as a promising tool for thermal-stability studies for proteins of therapeutic interest.

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“…CIU method is often used to study the structure change in protein . In this report, the effects of DMF with different concentrations on apo‐SOD1 conformation stability were assessed by CIU.…”

Section: Resultsmentioning

confidence: 99%

Effects of aprotic solvents on the stability of metal‐free superoxide dismutase probed by native electrospray ionization–ion mobility–mass spectrometry

et al. 2019

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Considering that aprotic solvents are often used as cosolvents in investigating the interactions between small molecules and proteins, we assessed the effects of five aprotic solvents represented by dimethylformamide (DMF) on the structure stabilities of metal‐free SOD1 (apo‐SOD1) by native electrospray ionization–ion mobility–mass spectrometry (ESI‐IM‐MS). These aprotic solvents include DMF, 1,3‐dimethyl‐2‐imidazolidinone (DMI), dimethyl sulfoxide (DMSO), acetonitrile (ACN), and tetrahydrofuran (THF). Results indicated that DMI, DMSO, and DMF at low percentage concentration could reduce the average charge and the dimer dissociation of apo‐SOD1. By contrast, ACN and THF at low concentration have no similar effect. DMF was selected as a representative solvent to further investigate the detailed effects on the structure stability of apo‐SOD1 by using collision‐induced dissociation and unfolding. The results reveal that the addition of minimal DMF to an aqueous protein solution can protect against the unfolding and dissociation of dimer, even under destabilizing conditions (such as low pH or high cone voltage). When the different percentage concentrations of DMF were added, the average collision cross section of apo‐SOD1 showed that apo‐SOD1 became compacted when the DMF concentration increased from 0% to 1% and eventually started extending when increased from 1% to 20%. The results indicated that DMF has similar effects to DMSO in native mass spectrometry (MS) and it can also be used as a cosolvent besides DMSO in investigating the stabilities of proteins and the interactions between small molecules and proteins.

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The effect of salt on the conformations of three model proteins is revealed by variable temperature ion mobility mass spectrometry

Cited by 26 publications

References 55 publications

Insights into the Conformations of Three Structurally Diverse Proteins: Cytochrome c, p53, and MDM2, Provided by Variable-Temperature Ion Mobility Mass Spectrometry

Insights into the Conformations of Three Structurally Diverse Proteins: Cytochrome c, p53, and MDM2, Provided by Variable-Temperature Ion Mobility Mass Spectrometry

Molecular Insights into the Thermal Stability of mAbs with Variable‐Temperature Ion‐Mobility Mass Spectrometry

Effects of aprotic solvents on the stability of metal‐free superoxide dismutase probed by native electrospray ionization–ion mobility–mass spectrometry

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