Quantitative prospective and retrospective mass spectrometry of lactoyl-CoA in mammalian cells and tissues

Varner, E. L.; Trefely, S.; Bartee, D.; von Krusenstiern, E.; Izzo, L.; Bekeova, C.; O'Connor, R. S.; Seifert, E. L.; Wellen, K.; Meier, J. L.; Snyder, N. W.

doi:10.1101/2020.03.15.992859

bioRxiv · 10.1101/2020.03.15.992859

Quantitative prospective and retrospective mass spectrometry of lactoyl-CoA in mammalian cells and tissues

Varner, E. L.·Trefely, S.·Bartee, D.·von Krusenstiern, E.·Izzo, L.·Bekeova, C.·O'Connor, R. S.·Seifert, E. L.·Wellen, K.·Meier, J. L.·Snyder, N. W.

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Abstract

Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalyzed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here we use liquid chromatography-high resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analyzed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate 13C3 15N1 -isotopically-labeled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14x10-8 pmol/cell in cell culture and 0.0172 pmol/mg tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20-350 times lower than predominant acyl-CoAs such as acetyl-, propionyl-, and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease. Highlights- Detection of lactoyl-CoA at picomole concentrations across tissues and cells - Lactoyl-CoA was detected at concentrations similar to crontonyl-CoA within HepG2 cells - Isotopically labeled 13C315N1-lactoyl-CoA can be prepared by SILEC

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Varner, E. L., Trefely, S., Bartee, D., von Krusenstiern, E., Izzo, L., Bekeova, C., O'Connor, R. S., Seifert, E. L., Wellen, K., Meier, J. L., Snyder, N. W.. 2020-03-17. Quantitative prospective and retrospective mass spectrometry of lactoyl-CoA in mammalian cells and tissues. https://doi.org/10.1101/2020.03.15.992859

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