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

Abstract

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.

The Ims Paradox: A Perspective on Structural Ion Mobility‐mass Spectrometry

“…At ~85°C the folded apoprotein dominates and the IM‐MS data reveal evidence (shift in the CCS profile and a mass loss of 2 Da) of the formation of a non‐native disulfide bond at high temperatures. The T m values obtained by VT‐ESI are in excellent agreement with those obtained by CD spectroscopy (64.5 and 67.0°C at λ 222nm and λ 209nm , respectively) (Woodall et al, 2019). This example illustrates the increased chemical information obtained from VT‐ESI coupled with IM‐MS: (i) the thermally induced loss of oxygen and the cofactor were not observed using CD spectroscopy, and (ii) the stabilization of the final product results from the formation of a non‐native disulfide bond.…”

“…( B ) Structures of the products formed by melting are shown along with respective CCS profiles and MS spectra. Reproduced from Woodall et al (2019). [Color figure can be viewed at wileyonlinelibrary.com]…”

A stochastic dynamic mass spectrometric diffusion method and its application to 3D structural analysis of the analytes

“…Because of this, as has been shown from our research, so far, the D SD parameter accounts for the multidimensional molecular conformation and electronic structure of the analyte ion. We may add that even with the employment of some of the most prominent MS methods, for instance, the ion mobility mass spectrometry, where the method operates within the spans of the measurement times between minutes and μs to ms, the MS structural information is mainly limited to 2D analysis as has been shown from the accumulated research more recently (Fuller et al, 2018;Beveridge et al, 2019;Chen et al, 2019;Woodall et al, 2019). The methodological efforts consisting of the computation of collision cross section (CCS) yields, in fact, to energetics of analyte ions, presumably corresponding to a 3D molecular conformation, which is con-nected with the drift time and the velocity of the MS ions via the Mason-Schamp equation.…”

Temperature‐Controlled Electrospray Ionization: Recent Progress and Applications

“…The latter cannot be examined by most other biophysical techniques use to study thermodynamics This research, in conjunction with similar work, set the stage for applications to more complex systems. [7,9,[74][75][76] Examples of these include utilizing ion mobility spectrometry (IMS) to investigate the of transition states of proteins during melting experiments, [6,56,57,61,77] investigations of the thermodynamics of ligands like lipids [78] or metal co-factors binding to biomolecules [61,79] , temperature-induced conformational transitions of antibodies, [36,80] structural transitions during oligomeric (C) Mass spectra representing dissociation of a wheat heat shock protein, TaHSP16.9, using TC-nESI-MS. Spectra at different temperatures show the dissociation on the oligomeric species (dodecamer, ~6000 m/z,) into suboligomeric species (at 1000 -4000 m/z). Selected charge states for monomers (solid type) and dimers (outlined type) are labelled.…”

“…[82] An example of how IMS can be combined with temperature-controlled ESI can be seen in the study . [79] In this work, TC-ESI-MS was coupled with IMS to elucidate the conformational changes myohemerythrin (Mhr) during thermal denaturation. Eighteen unique conformational intermediates were detected for this protein.…”