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It has been challenging to elucidate cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live\u2010cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ\u2013synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analogue cofactors can only be processed by the engineered PMTs\u2014and not native PMTs\u2014to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome\u2010wide methylation and validate specific methylation events. \u00a9 2021 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Live\u2010cell labeling of substrates of protein methyltransferases GLP1 and PRMT1 with lcBPPM\u2010feasible enzymes and SAM analogue precursors<\/jats:p><jats:p><jats:bold>Support Protocol<\/jats:bold>: Gram\u2010scale synthesis of Hey\u2010Met<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Click labeling of lcBPPM cell lysates with a biotin\u2010azide probe<\/jats:p><jats:p><jats:bold>Alternate Protocol<\/jats:bold>: Click labeling of small\u2010scale lcBPPM cell lysates with a TAMRA\u2010azide dye for in\u2010gel fluorescence visualization<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Enrichment of biotinylated lcBPPM proteome with streptavidin beads<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Proteome\u2010wide identification of lcBPPM targets with mass spectrometry<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Validation of individual lcBPPM targets by western blot<\/jats:p>","DOI":"10.1002\/cpz1.213","type":"journal-article","created":{"date-parts":[[2021,8,9]],"date-time":"2021-08-09T20:06:58Z","timestamp":1628539618000},"source":"Crossref","is-referenced-by-count":4,"title":["Profiling and Validation of Live\u2010Cell Protein Methylation with Engineered Enzymes and Methionine Analogues"],"prefix":"10.1002","volume":"1","author":[{"given":"Nicole","family":"Weiss","sequence":"first","affiliation":[{"name":"BCMB Allied Program Weill Cornell Medical College, Cornell University  New York New York"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chamara","family":"Seneviranthe","sequence":"additional","affiliation":[{"name":"Chemical Biology Program Memorial Sloan Kettering Cancer Center  New York New 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