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Online protein unfolding characterized by ion mobility electron capture dissociation mass spectrometry: cytochrome C from neutral and acidic solutions

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Abstract

Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation (ECD) of proteins ionized from aqueous solutions, have been used for the study of solution-like structures of intact proteins. By mixing aqueous proteins with denaturants online before ESI, the amount of protein unfolding can be precisely controlled and rapidly analyzed, permitting the characterization of protein folding intermediates in protein folding pathways. Herein, we mixed various pH solutions online with aqueous cytochrome C for unfolding and characterizing its unfolding intermediates with ESI-MS charge state distribution measurements, IMS, and ECD. The presence of folding intermediates and unfolded cytochrome c structures were detected from changes in charge states, arrival time distributions (ATDs), and ECD. We also compared structures from nondenaturing and denaturing solution mixtures measured under “gentle” (i.e., low energy) ion transmission conditions with structures measured under “harsh” (i.e., higher energy) transmission. This work confirms that when using “gentle” instrument conditions, the gas-phase cytochrome c ions reflect attributes of the various solution-phase structures. However, “harsh” conditions that maximize ion transmission produce extended structures that no longer correlate with changes in solution structure.

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Acknowledgements

The authors would like to acknowledge Professor Derek Wilson and his research group for helpful discussions.

Funding

Portions of this work were funded by the National Science Foundation CAREER CHE-2143755.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Indiana University–Purdue University Indianapolis, Indianapolis, IN, 46202, USA

    Rebecca L. Cain & Ian K. Webb

  2. Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

    Ian K. Webb

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  1. Rebecca L. Cain
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Cain, R.L., Webb, I.K. Online protein unfolding characterized by ion mobility electron capture dissociation mass spectrometry: cytochrome C from neutral and acidic solutions. Anal Bioanal Chem 415, 749–758 (2023). https://doi.org/10.1007/s00216-022-04501-w

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