{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T05:38:28Z","timestamp":1762234708666,"version":"build-2065373602"},"reference-count":38,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T00:00:00Z","timestamp":1761868800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Science Foundation","award":["2203942","1808087"],"award-info":[{"award-number":["2203942","1808087"]}]},{"name":"National Institute of General Medical Sciences of the National Institute of Health","award":["R35GM156318"],"award-info":[{"award-number":["R35GM156318"]}]},{"name":"UGA Dissertation Completion Award"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Methacrylyl-CoA is a key metabolic intermediate in the valine catabolic pathway. Its accumulation has been found to be cytotoxic and associated with pathological conditions. Nevertheless, detailed biological effects of methacrylyl-CoA and methacrylate in human physiology and pathology are poorly understood. We propose that the electrophilicity of the alkene bond in the methacrylyl group can react with the cysteine residues in proteins resulting in an unexplored protein post-translational modification (PTM), cysteine S-2-carboxypropylation (C2cp). To test and validate this mechanistic hypothesis, we experimentally detected and profiled S-2-carboxypropylated proteins from the complex cellular proteome with the design and application of a bioorthogonal chemical probe, N-propargyl methacrylamide. We tested the probe in different mammalian cell models and demonstrated its versatility and sensitivity to protein cysteine S-2-carboxypropylation. We established quantitative chemical proteomics for global and site-specific profiling of protein S-2-carboxypropylation, which successfully identified 403 S-2-carboxypropylated proteins and 120 cysteine modification sites from HEK293T cells. Through bioinformatic analysis, we found that C2cp-modified proteins were involved in a variety of critical cellular functions including translation, RNA splicing, and protein folding. Our chemoproteomic studies demonstrating the proteome-wide distribution of cysteine S-2-carboxypropylation provide a new biochemical mechanism for the functional investigation of methacrylyl-CoA and understanding valine-related metabolic disorders.<\/jats:p>","DOI":"10.3390\/molecules30214255","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T16:18:42Z","timestamp":1762186722000},"page":"4255","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Discovery and Profiling of Protein Cysteine S-2-Carboxypropylation"],"prefix":"10.3390","volume":"30","author":[{"given":"Jiabao","family":"Song","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kejun","family":"Yin","sequence":"additional","affiliation":[{"name":"School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ronghu","family":"Wu","sequence":"additional","affiliation":[{"name":"School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7116-3067","authenticated-orcid":false,"given":"Y. George","family":"Zheng","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1547S","DOI":"10.1093\/jn\/135.6.1547S","article-title":"Hormonal and signaling role of branched-chain amino acids","volume":"135","author":"Nair","year":"2005","journal-title":"J. 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