{"doi":"10.1101/2020.03.15.992859","title":"Quantification of lactoyl-CoA by liquid chromatography mass spectrometry in mammalian cells and tissues","abstract":"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 13 C 3 15 N 1 -isotopically-labeled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14×10 −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 13 C 3 15 N 1 -lactoyl-CoA can be prepared by SILEC","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":126398,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9537,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":237763,"name":"Sophie Trefely","orcid":"0000-0003-3816-6869","position":1,"is_corresponding":false},{"id":260635,"name":"David Bartee","orcid":"0000-0001-8090-4242","position":2,"is_corresponding":false},{"id":260636,"name":"Eliana von Krusenstiern","orcid":"0000-0002-9569-3262","position":3,"is_corresponding":false},{"id":260637,"name":"Luke Izzo","orcid":"0000-0002-6217-9240","position":4,"is_corresponding":false},{"id":574323,"name":"Carmen Bekeova","orcid":null,"position":5,"is_corresponding":false},{"id":574324,"name":"Roddy S O’Connor","orcid":null,"position":6,"is_corresponding":false},{"id":261603,"name":"Erin L. Seifert","orcid":null,"position":7,"is_corresponding":false},{"id":574325,"name":"Kathryn Wellen","orcid":null,"position":8,"is_corresponding":false},{"id":120865,"name":"Jordan L. Meier","orcid":null,"position":9,"is_corresponding":false},{"id":237765,"name":"Nathaniel W. Snyder","orcid":"0000-0001-5643-8747","position":10,"is_corresponding":false},{"id":574322,"name":"Erika L Varner","orcid":null,"position":0,"is_corresponding":true}],"reference_count":17,"raw_metadata":null,"created_at":"2026-07-18T23:15:23.509897Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}