A comparison of mass spectrometry based hydrogen deuterium exchange methods for probing the cyclophilin A cyclosporin complex

Esswein, Stefan T.; Florance, Hannah; Baillie, Laura; Lippens, Jessica; Barran, Perdita E.

doi:10.1016/j.chroma.2010.05.028

Cited by 12 publications

(10 citation statements)

References 43 publications

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“…In 20 mM ammonium acetate with pH = 6.8, charge states as low as +7 can be observed, consistent with the work by Barran and co-workers. 66 The plot of CCSs versus charge state (Figure 8B) and comparison to theoretical CCSs of CypA X-ray structures (PDB IDs 5LUD, 67 1AWR, 68 and 1AWV 68 ) show that the protein retains its native state at z = +7 and +8. The large increase seen in charge states from z = +8 to z = +9 indicates the unfolding of the native CypA structure as extra charge states accrue, a phenomenon that is well documented for many proteins.…”

Section: Our Data Indicate That the Long-established View On The Comp...mentioning

confidence: 95%

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis–Trans Isomerization and the Chameleonic Roles of Calcium

et al. 2021

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Falling outside of Lipinski’s rule of five, macrocyclic drugs have accessed unique binding sites of their target receptors unreachable by traditional small molecules. Cyclosporin(e) A (CycA), an extensively studied macrocyclic natural product, is an immunosuppressant with undesirable side effects such as electrolytic imbalances. In this work, a comprehensive view on the conformational landscape of CycA, its interactions with Ca2+, and host–guest interactions with cyclophilin A (CypA) is reported through exhaustive analyses that combine ion-mobility spectrometry–mass spectrometry (IMS–MS), nuclear magnetic resonance (NMR) spectroscopy, distance-geometry modeling, and NMR-driven molecular dynamics. Our IMS–MS data show that CycA can adopt extremely compact conformations with significantly smaller collisional cross sections than the closed conformation observed in CDCl3. To adopt these conformations, the macrocyclic ring has to twist and bend via cis–trans isomerization of backbone amides, and thus, we termed this family of structures the “bent” conformation. Furthermore, NMR measurements indicate that the closed conformation exists at 19% in CD3OD/H2O and 55% in CD3CN. However, upon interacting with Ca2+, in addition to the bent and previously reported closed conformations of free CycA, the CycA:Ca2+ complex is open and has all-trans peptide bonds. Previous NMR studies using calcium perchlorate reported only the closed conformation of CycA (which contains one cis peptide bond). Here, calcium chloride, a more biologically relevant salt, was used, and interestingly, it helps converting the cis -MeLeu9–MeLeu10 peptide bond into a trans bond. Last, we were able to capture the native binding of CycA and CypA to give forth evidence that IMS–MS is able to probe the solution-phase structures of the complexes and that the Ca2+:CycA complex may play an essential role in the binding of CycA to CypA.

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Section: Our Data Indicate That the Long-established View On The Comp...mentioning

confidence: 95%

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis–Trans Isomerization and the Chameleonic Roles of Calcium

et al. 2021

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“…27 Exposure of Melittin to deuterated methanol in the ion trap at times greater than 10000 ms was desirable to elicit more information regarding analyte stability in the gas phase. 27 Exposure of Melittin to deuterated methanol in the ion trap at times greater than 10000 ms was desirable to elicit more information regarding analyte stability in the gas phase.…”

Section: Hdx Mass Spectrometrymentioning

confidence: 99%

“…Experiments were performed on melittin sprayed from a range of solvents using nanoESI on an LCQ (Thermo Scientific USA) with a modified helium inlet line, which incorporates a reservoir for deuterated solvent. 27 Exposure of Melittin to deuterated methanol in the ion trap at times greater than 10000 ms was desirable to elicit more information regarding analyte stability in the gas phase. To this end a software patch specifying an extended activation time command of 100 s was supplied by Thermo Scientific.…”

Section: Hdx Mass Spectrometrymentioning

confidence: 99%

Evidence for α-helices in the gas phase: A case study using Melittin from honey bee venom

Florance

Stopford

Kalapothakis

et al. 2011

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Gas phase methodologies are increasingly used to study the structure of proteins and peptides. A challenge to the mass spectrometrist is to preserve the structure of the system of interest intact and unaltered from solution into the gas phase. Small peptides are very flexible and can present a number of conformations in solution. In this work we examine Melittin a 26 amino acid peptide that forms the active component of honey bee venom. Melittin is haemolytic and has been shown to form an α-helical tetrameric structure by X-ray crystallography [M. Gribskov et al., The RCSB Protein Data Bank, 1990] and to be helical in high concentrations of methanol. Here we use ion mobility mass spectrometry, molecular dynamics and gas-phase HDX to probe its structure in the gas phase and specifically interrogate whether the helical form can be preserved. All low energy calculated structures possess some helicity. In our experiments we examine the peptide following nano-ESI from solutions with varying methanol content. Ion mobility gives collision cross sections (CCS) that compare well with values found from molecular modelling and from other reported structures, but with inconclusive results regarding the effect of solvent. There is only a slight increase in CCS with charge, showing minimal coloumbically driven unfolding. HDX supports preservation of some helical content into the gas phase and again shows little difference in the exchange rates of species sprayed from different solvents. The [M + 3H](3+) species has two exchanging populations both of which exhibit faster exchange rates than observed for the [M + 2H](2+) species. One interpretation for these results is that the time spent being analysed is sufficient for this peptide to form a helix in the 'ultimate' hydrophobic environment of a vacuum.

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“…H/D exchange in ESI‐MS was used in order to determine the number and position of exchangeable hydrogen atoms of pharmaceuticals and peptides, to characterize metabolites by determination of exchangeable hydrogen atoms, for studying protein‐ligand interactions, for conformational studies, and for elucidation of fragmentation pathways . Besides application of deuterated mobile phases, the exchange was achieved by post‐column infusion of D 2 O, by introducing the deuterating agent (e.g., MeOD) into the curtain gas flow of a commercial triple‐quadrupole mass spectrometer, by using D 2 O as a sheath liquid, or by application of a dual‐sprayer atmospheric pressure ionization source with one spraying D 2 O …”

mentioning

confidence: 99%

Isomerization of cannabidiol and Δ⁹‐tetrahydrocannabinol during positive electrospray ionization. In‐source hydrogen/deuterium exchange experiments by flow injection hybrid quadrupole‐time‐of‐flight mass spectrometry

Broecker

Pragst

2012

Rapid Comm Mass Spectrometry

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Injection of the investigated substances in D(2) O and measurement by FI-QTOF-MS proved to be an efficient way to perform H/D exchange experiments. The results were interpreted as an acid-catalyzed in-source equilibration between THC and CBD leading to the same precursor ions and to an H/D exchange in the methyl groups under the increased acidic conditions in the positive ESI droplets. Therefore, in positive LC/ESI-MS/MS, peak identification by CID spectra or by abundance ratio of multiple reaction monitoring (MRM) transitions is not sufficient for unambiguous discrimination between THC and CBD and must be supported by retention time or other experimental evidence.

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A comparison of mass spectrometry based hydrogen deuterium exchange methods for probing the cyclophilin A cyclosporin complex

Cited by 12 publications

References 43 publications

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis–Trans Isomerization and the Chameleonic Roles of Calcium

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis–Trans Isomerization and the Chameleonic Roles of Calcium

Evidence for α-helices in the gas phase: A case study using Melittin from honey bee venom

Isomerization of cannabidiol and Δ⁹‐tetrahydrocannabinol during positive electrospray ionization. In‐source hydrogen/deuterium exchange experiments by flow injection hybrid quadrupole‐time‐of‐flight mass spectrometry

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A comparison of mass spectrometry based hydrogen deuterium exchange methods for probing the cyclophilin A cyclosporin complex

Cited by 12 publications

References 43 publications

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis–Trans Isomerization and the Chameleonic Roles of Calcium

Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis–Trans Isomerization and the Chameleonic Roles of Calcium

Evidence for α-helices in the gas phase: A case study using Melittin from honey bee venom

Isomerization of cannabidiol and Δ9‐tetrahydrocannabinol during positive electrospray ionization. In‐source hydrogen/deuterium exchange experiments by flow injection hybrid quadrupole‐time‐of‐flight mass spectrometry

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Isomerization of cannabidiol and Δ⁹‐tetrahydrocannabinol during positive electrospray ionization. In‐source hydrogen/deuterium exchange experiments by flow injection hybrid quadrupole‐time‐of‐flight mass spectrometry