Mass Spectrometry-based Lactylome and Confirmation of Lactylated Proteins

First identified in 2019, lactylation, a new post-translational modification (PTM) on lysine residues, has since been shown to be of interest in multiple pathologies and physiological contexts. It possesses two isomers (L- and D-lactylation) and is known to alter complex formation, cellular localization, or stability of target proteins. This protocol describes a method for analyzing lactylation from cell culture to confirmation of potential lactylated targets, using the TE11 esophageal squamous cell carcinoma cell line as an example. The following protocol will provide details on the identification of lactylated proteins through L-lactyllysine (KL-LA)-specific immunoprecipitation (IP) of peptides for mass spectrometry (MS) analysis: (1) protein extraction, (2) protein reduction, alkylation and digestion, (3) protein purification and peptides concentration, (4) IP using KL-LA beads and (5) concentration of KL-LA peptides. Following these steps, analyses of the MS raw data will be performed to identify lactylated sites, and confirmation of these putative lactylated proteins by IP will be described. Using this method, a range of 100 to 500 peptides can be identified, and lactylated proteins of interest can be confirmed. To conclude, studying lactylated proteins has the potential to enhance our understanding of PTM-driven cell signaling in both normal and disease conditions.