Abstract
<jats:p>While medium-chain fatty acids (MCFAs; C6-C12) are key metabolites with profound physiological roles, their potential to covalently modify proteins remains an open question, with octanoylation of only one protein documented to date. Here, we report the discovery of three novel MCFA modifications, including hexanoylation (C6), decanoylation (C10), and dodecanoylation (C12), and present the first proteome-wide analysis of medium-chain acylation. Using chemo-proteomic methods, we identified hundreds of protein substrates for each modification, including 270 hexanoylated, 131 octanoylated, 286 decanoylated, and 509 dodecanoylated proteins. We further validated cysteine hexanoylation by mass spectrometry in FAHD2A, a mitochondrial oxaloacetate isomerase highly enriched among the 270 hexanoylated proteins, and identified the putative “writer” and “eraser” enzymes, acyltransferase ZDHHC3 and deacylase SIRT4, respectively. Our discovery of medium-chain acylation significantly expands the landscape of protein lipidation, and the proteomic atlas of medium-chain acylation provides a foundational resource for elucidating the mechanisms underlying the MCFA’s physiological functions.</jats:p>