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bioRxiv · 10.1101/2022.07.22.501130

Dynamic in vivo mapping of the methylproteome using a chemoenzymatic approach

Farhi, J.·Jones, R. B.·Verma, A. K.·Parwani, K. K.·Emenike, B.·Tang, K.-C.·Bagchi, P.·Raj, M.·Spangle, J.
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Abstract

Dynamic protein post-translation methylation is essential for cellular function, highlighted by the essential role of methylation in transcriptional regulation and its aberrant dysregulation in diseases including cancer. This underscores the importance of cataloging the cellular methylproteome. However, comprehensive analysis of the methylproteome remains elusive due to limitations in current enrichment and analysis pipelines. Here, we employ an L-Methionine analogue, ProSeMet, that is chemoenzymatically converted to the SAM analogue ProSeAM in cells and in vivo to tag proteins with a biorthogonal alkyne that can be directly detected via LC-MS/MS, or functionalized for subsequent selective enrichment and LC-MS/MS identification. Without enrichment, we identify lysine mono-, di-, and trimethylation, histidine methylation, and arginine methylation with site specific resolution on proteins including heat shock protein HSPA8, for which methylation is implicated in human disease. With enrichment, we identify 486 proteins known to be methylated and 221 proteins with novel methylation sites encompassing diverse cellular functions. Systemic ProSeMet delivery in mice pseudomethylates proteins across organ systems with blood-brain barrier penetrance and identifies site-specific pseudomethylation in vivo with LC-MS/MS. Leveraging these pipelines to define the cellular methylproteome may have broad applications for understanding the methylproteome in the context of disease.

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Farhi, J., Jones, R. B., Verma, A. K., Parwani, K. K., Emenike, B., Tang, K.-C., Bagchi, P., Raj, M., Spangle, J.. 2022-07-22. Dynamic in vivo mapping of the methylproteome using a chemoenzymatic approach. https://doi.org/10.1101/2022.07.22.501130

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