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Chemoproteomic profiling and discovery of protein electrophiles in human cells

Nature Chemistry volume 9, pages 234–243 (2017)Cite this article

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Abstract

Activity-based protein profiling (ABPP) serves as a chemical proteomic platform to discover and characterize functional amino acids in proteins on the basis of their enhanced reactivity towards small-molecule probes. This approach, to date, has mainly targeted nucleophilic functional groups, such as the side chains of serine and cysteine, using electrophilic probes. Here we show that ‘reverse-polarity’ (RP)-ABPP using clickable, nucleophilic hydrazine probes can capture and identify protein-bound electrophiles in cells. Using this approach, we demonstrate that the pyruvoyl cofactor of S-adenosyl-L-methionine decarboxylase (AMD1) is dynamically controlled by intracellular methionine concentrations. We also identify a heretofore unknown modification—an N-terminally bound glyoxylyl group—in the poorly characterized protein secernin-3. RP-ABPP thus provides a versatile method to monitor the metabolic regulation of electrophilic cofactors and discover novel types of electrophilic modifications on proteins in human cells.

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Figure 1: RP-ABPP with hydrazine probes in human cells.
Figure 2: Identification of protein targets of hydrazine probes.
Figure 3: Functional profiling of the pyruvoyl cofactor of AMD1 by hydrazine probes.
Figure 4: Identification of a hydrazine-reactive site in SCRN3.
Figure 5: Evidence supporting the structural assignment of an N-terminal glyoxylyl group in SCRN3.
Figure 6: Possible routes for N-terminal processing of SCRN3 in comparison to the established mechanisms of N-terminal maturation for other protein classes.

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Acknowledgements

We acknowledge Steven Ealick and Leslie Kinsland for the AdoMetDC bacterial expression plasmid, Keriann Backus for the cleavable tags, Gonzalo González-Páez and Dennis Wolan for TEV protease, Melissa Dix and Jim Moresco for instrument assistance, Armand Cognetta and Kenneth Lum for software and data assistance, Liron Bar-Peled and Stephan Hacker for plasmids and Silvia Ortega-Gutiérrez for helpful discussions. This work was supported by the National Institutes of Health Grants CA132630 (B.F.C.), P41 GM103533 and U54 GM114833-02 (J.R.Y.), an NSF Graduate Research Fellowship DGE-1346837 (E.J.O.) and a Helen Hay Whitney Postdoctoral Fellowship sponsored by Merck (M.L.M).

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Authors and Affiliations

  1. Chemical Physiology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Megan L. Matthews, Lin He, Benjamin D. Horning, Bruno E. Correia, John R. Yates III & Benjamin F. Cravatt

  2. Bioinformatics Solutions Inc, Waterloo, N2L 6J2, Ontario, Canada

    Lin He

  3. Chemistry and Cell and Molecular Biology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Erika J. Olson & Philip E. Dawson

  4. École polytechnique fédérale de Lausanne, Lausanne, CH-1015, Switzerland

    Bruno E. Correia

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Contributions

M.L.M and B.F.C. conceived the project, designed experiments and composed the manuscript. M.L.M performed all experiments. L.H. wrote software and J.R.Y. provided advice. M.L.M., L.H., B.E.C. and B.F.C. analysed data. M.L.M., E.J.O. and P.E.D. designed peptide standards. M.L.M. and E.J.O. synthesized peptides. M.L.M. and B.D.H. synthesized probes.

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Correspondence to Megan L. Matthews or Benjamin F. Cravatt.

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Matthews, M., He, L., Horning, B. et al. Chemoproteomic profiling and discovery of protein electrophiles in human cells. Nature Chem 9, 234–243 (2017). https://doi.org/10.1038/nchem.2645

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