Quantification of lactoyl-CoA (lactyl-CoA) by liquid chromatography mass spectrometry in mammalian cells and tissues

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-catalysed 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 analysed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate ¹³C₃¹⁵N₁-isotopically labelled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14 × 10^-8 pmol per cell in cell culture and 0.0172 pmol mg^-1 tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20 and 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 in metazoans, and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease.

Keywords: LC-HRMS; high resolution; lactoyl-CoA; lactyl-CoA; metabolism.

Figure 1.

(a) Structure of lactoyl-CoA (stereochemistry not shown). (b) LC-HRMS of synthetic lactoyl-CoA. (c) LC-MS/HRMS of lactoyl-CoA.

Figure 2.

(a) LC-HRMS of lactoyl-CoA from HepG2 cell extract and (b) LC-MS/HRMS of synthetic lactoyl-CoA (left) and the same ions in cell extract (right).

Figure 3.

Detection of lactoyl-CoA from retrospective analysis (a) of tissues by LC-HRMS (top) and LC-MS/HRMS (bottom) and (b) co-elution of lactoyl-CoA with ¹³C₃¹⁵N₁-lactoyl-CoA from retrospective data.