Session: Parallel session 4 - PTM
Characterization of histone lysine lactylation in mouse testis
In the nucleus of eukaryotes, DNA wraps around octamers of histone proteins to form a structure called chromatin. Post-translational modifications (PTMs) of histones play a crucial role in regulating gene expression. One well-studied modification occurring on lysine residues is acetylation, which is associated with chromatin accessibility and active gene transcription. Since its discovery in 2019, histone lysine lactylation has been widely studied in many contexts for its role in the metabolic regulation of gene expression. We have undertaken the task of characterizing this PTM in mouse spermatogenesis by proteomics.
We used mass spectrometry Data-Dependent Acquisition (DDA) and targeted proteomics to characterize histone lactylation compared to acetylation in mouse testis. More specifically, because both L-lactyl and D-lactyl could be anticipated, we used isotope-labeled synthetic peptides corresponding to both enantiomers to perform analyses by Parallel Reaction Monitoring.
First, by DDA, we were able to map lactylation and acetylation on many lysines of histones H3 and H4. We carefully validated all identifications by checking the presence in MS/MS spectra of diagnostic ions corresponding to acetylated or lactylated lysines in the fragmented peptides. The quantification of lactylation was tedious because of oxidation events and it led us to perform targeted proteomics. Peptides bearing L-lactyl or D-lactyl could be often but not always separated by chromatography. Our results showed that L-lactyl and D-lactyl were of similar stoichiometry on most lysines of H3 and H4.
In conclusion, we implemented a meticulous analysis to distinguish L-lactylated from D-lactylated lysines on histones from mouse testis. Our results enable to hypothesize the involvement of the glyoxalase DJ-1 in producing a racemic mixture of L- and D-lactate from methylglyoxal in the chromatin environment, to serve as a substrate for histone lactylation.
We used mass spectrometry Data-Dependent Acquisition (DDA) and targeted proteomics to characterize histone lactylation compared to acetylation in mouse testis. More specifically, because both L-lactyl and D-lactyl could be anticipated, we used isotope-labeled synthetic peptides corresponding to both enantiomers to perform analyses by Parallel Reaction Monitoring.
First, by DDA, we were able to map lactylation and acetylation on many lysines of histones H3 and H4. We carefully validated all identifications by checking the presence in MS/MS spectra of diagnostic ions corresponding to acetylated or lactylated lysines in the fragmented peptides. The quantification of lactylation was tedious because of oxidation events and it led us to perform targeted proteomics. Peptides bearing L-lactyl or D-lactyl could be often but not always separated by chromatography. Our results showed that L-lactyl and D-lactyl were of similar stoichiometry on most lysines of H3 and H4.
In conclusion, we implemented a meticulous analysis to distinguish L-lactylated from D-lactylated lysines on histones from mouse testis. Our results enable to hypothesize the involvement of the glyoxalase DJ-1 in producing a racemic mixture of L- and D-lactate from methylglyoxal in the chromatin environment, to serve as a substrate for histone lactylation.