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Irregularities in kinase activity are directly associated with disease conditions. Therefore, methods to identify substrates of a particular kinase are needed to understand signaling cascades in normal and diseased states. Photocrosslinking ATP analogs provide powerful tools to study kinases by covalently linking kinases with substrates. However, the involvement of UV light and nonspecific reactivity of current ATP\u2010photocrosslinkers challenge kinase\u2013substrate identification. We report here an affinity\u2010based crosslinking ATP analog, ATP\u2010methylacrylamide (ATP\u2010MAc), that contains a cysteine\u2010reactive acrylamide crosslinking group, which avoids the UV irradiation and non\u2010specific reactivity of prior analogs. Using in\u2005vitro kinase assays, ATP\u2010MAc acts as a kinase co\u2010substrate and covalently crosslinks only kinases containing cysteines in the active site. ATP\u2010MAc was also able to crosslink cellular proteins in lysates, documenting compatibility with cell\u2010based studies.<\/jats:p>","DOI":"10.1002\/anie.202014047","type":"journal-article","created":{"date-parts":[[2021,2,23]],"date-time":"2021-02-23T03:11:47Z","timestamp":1614049907000},"page":"9859-9862","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["An Affinity\u2010Based, Cysteine\u2010Specific ATP Analog for Kinase\u2010Catalyzed Crosslinking"],"prefix":"10.1002","volume":"60","author":[{"given":"Ahmed E.","family":"Fouda","sequence":"first","affiliation":[{"name":"Department of Chemistry Wayne State University  5101, Cass Ave Detroit MI 48202 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aparni K.","family":"Gamage","sequence":"additional","affiliation":[{"name":"Department of Chemistry Wayne State University  5101, Cass Ave Detroit MI 48202 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1396-3194","authenticated-orcid":false,"given":"Mary Kay H.","family":"Pflum","sequence":"additional","affiliation":[{"name":"Department of Chemistry Wayne State University  5101, Cass Ave Detroit MI 48202 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2021,3,23]]},"reference":[{"key":"e_1_2_2_1_1","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(95)90405-0"},{"key":"e_1_2_2_2_1","unstructured":"\u00a0"},{"key":"e_1_2_2_3_2","doi-asserted-by":"publisher","DOI":"10.1021\/cr000230w"},{"key":"e_1_2_2_4_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.48.070179.004423"},{"key":"e_1_2_2_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/cb300610s"},{"key":"e_1_2_2_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi991109q"},{"key":"e_1_2_2_7_1","unstructured":"\u00a0"},{"key":"e_1_2_2_8_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.94.8.3565"},{"key":"e_1_2_2_9_2","doi-asserted-by":"publisher","DOI":"10.1021\/ja050727t"},{"key":"e_1_2_2_10_2","doi-asserted-by":"publisher","DOI":"10.1021\/ja048659i"},{"key":"e_1_2_2_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0014-5793(02)02778-3"},{"key":"e_1_2_2_12_1","unstructured":"\u00a0"},{"key":"e_1_2_2_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmc.2014.01.034"},{"key":"e_1_2_2_14_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200905244"},{"key":"e_1_2_2_14_3","doi-asserted-by":"publisher","DOI":"10.1002\/ange.200905244"},{"key":"e_1_2_2_15_1","unstructured":"\u00a0"},{"key":"e_1_2_2_16_2","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.8b10160"},{"key":"e_1_2_2_17_2","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.6b00289"},{"key":"e_1_2_2_18_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12953-017-0123-3"},{"key":"e_1_2_2_19_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.91"},{"key":"e_1_2_2_20_1","doi-asserted-by":"publisher","DOI":"10.1021\/jm101396q"},{"key":"e_1_2_2_21_1","doi-asserted-by":"publisher","DOI":"10.1021\/jm0005555"},{"key":"e_1_2_2_22_1","unstructured":"\u00a0"},{"key":"e_1_2_2_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2006.05.013"},{"key":"e_1_2_2_24_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M207135200"},{"key":"e_1_2_2_25_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja066828o"},{"key":"e_1_2_2_26_1","unstructured":"W.\u2005L. 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