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Here, we develop and experimentally benchmark a bioorthogonal probe platform for labeling, profiling, and imaging of lysine vitcylation, a recently identified vitamin C\u2010derived post\u2010translational modification, in living cells. Chemoproteomic analysis and fluorescence imaging reveal a candidate vitcylome enriched in nucleolar proteins associated with ribonucleoprotein biogenesis and RNA processing, and reporter\u2010derived lysine adducts on selected targets were validated by mass spectrometry. Time\u2010resolved analyses demonstrate that intracellular vitcylation undergoes unexpectedly rapid remodeling, indicating active cellular regulation. Furthermore, a genome\u2010wide CRISPR\u2010Cas9 screen identifies glutathione S\u2010transferase P1 (GSTP1) as an upstream regulator of global vitamin C\u2010derived protein modification, linking vitcylation remodeling to cellular electrophile handling and redox homeostasis. Collectively, this platform provides a chemical framework for profiling, visualizing, and investigating protein vitcylation in living cells, and reveals vitcylation as a dynamic, spatially organized and regulated metabolic modification.<\/jats:p>","DOI":"10.1002\/anie.2501228","type":"journal-article","created":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T05:41:54Z","timestamp":1791870114000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Bioorthogonal Vitamin C Probes Enable Cellular Profiling and Investigation of Lysine Vitcylation"],"prefix":"10.1002","author":[{"given":"Wanting","family":"Wang","sequence":"first","affiliation":[{"name":"Hubei Hongshan Laboratory College of Biomedicine and Health College of Life Science and Technology Huazhong Agricultural University  Wuhan 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Commission of the PR China Lifecycle Health Management Center Ruijin Hospital Shanghai Jiao Tong University School of Medicine  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8285-5795","authenticated-orcid":false,"given":"Cheng\u2010Liang","family":"Zhu","sequence":"additional","affiliation":[{"name":"College of Pharmaceutical Sciences Zhejiang University  Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5044-9947","authenticated-orcid":false,"given":"Shuai","family":"Zhang","sequence":"additional","affiliation":[{"name":"Hubei Hongshan Laboratory College of Biomedicine and Health College of Life Science and Technology Huazhong Agricultural University  Wuhan 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