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Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis–MS

Nature Chemical Biology volume 12, pages 918–922 (2016)Cite this article

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Abstract

To enable the detection of protein conformational isomers, their enzymatic activity and their inhibition in a single experiment, we developed a method based on kinetic capillary electrophoresis coupled on-line with UV detection and ion mobility mass spectrometry (CE–UV–IM–MS). Kinetic CE–UV separated protein conformers and monitored their interconversion dynamics in solution. Ion mobility mass spectrometry analyzed the conformer sizes, exact molecular weights, and structures of an enzyme and of its substrates, inhibitors and corresponding products. This coupled CE–UV–IM–MS system allowed the simultaneous, real-time observation of the effect of small-molecule inhibitors on both the conformational distribution and enzymatic activity of the human tissue transglutaminase TG2. By expanding mass spectrometry profiling of enzymatic reactions beyond proteins and substrates to include protein dynamics, CE–UV–IM–MS opens a new avenue for the modulation and regulation of cellular functions, drug development and protein engineering.

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Figure 1: Schematic representation of CE–UV–IM–MS method.
Figure 2: CE–UV–IM–MS experimental data.
Figure 3: Effects of inhibitors on enzyme structure and activity.

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Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada (M.V.B. and J.W.K.), the Canada Foundation for Innovation (M.V.B.), the Ministry of Research and Innovation, Ontario, Canada (M.V.B.) and by an Ontario Graduate Scholarship (for G.G.M.).

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Authors and Affiliations

  1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada

    Gleb G Mironov, Christopher M Clouthier, Abdullah Akbar, Jeffrey W Keillor & Maxim V Berezovski

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  1. Gleb G Mironov
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  2. Christopher M Clouthier
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Contributions

G.G.M., J.W.K., and M.V.B. conceived of the project, designed the experiments, analyzed the data, and wrote the manuscript. G.G.M. built the CE–UV–IM–MS system and performed all CE–UV, IM–MS and CE–UV–IM–MS experiments, conformational analysis, and inhibition analysis. C.M.C. expressed and purified TG2 protein, synthesized peptide substrates, and assisted in writing the manuscript; A.A. synthesized inhibitors, measured inhibition kinetics with a chromogenic substrate, and assisted in writing the manuscript.

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Correspondence to Jeffrey W Keillor or Maxim V Berezovski.

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The authors declare no competing financial interests.

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Mironov, G., Clouthier, C., Akbar, A. et al. Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis–MS. Nat Chem Biol 12, 918–922 (2016). https://doi.org/10.1038/nchembio.2170

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