{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T19:46:23Z","timestamp":1792784783669,"version":"4.5.0"},"reference-count":49,"publisher":"American Chemical Society (ACS)","issue":"9","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100012226","name":"Fundamental Research Funds for the Central Universities","doi-asserted-by":"publisher","award":["WK9990250190"],"award-info":[{"award-number":["WK9990250190"]}],"id":[{"id":"10.13039\/501100012226","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Natural Science Foundation of China (NSFC)","award":["22377117"],"award-info":[{"award-number":["22377117"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"crossref"}]},{"name":"National Natural Science Foundation of China (NSFC)","award":["22407120"],"award-info":[{"award-number":["22407120"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"crossref"}]},{"name":"National Natural Science Foundation of China (NSFC)","award":["22577128"],"award-info":[{"award-number":["22577128"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Activity-based ubiquitin probes have emerged as powerful tools for dissecting protein ubiquitination. In this work, we discovered that an \u03b1-amidomethyl-substituted acrylate electrophile exhibits markedly enhanced reactivity. Based on this finding, we designed and synthesized a new activity-based probe, ubiquitin-\u03b1-amidomethyl acrylate (Ub-AMA). In vitro biochemical assays demonstrated that Ub-AMA effectively crosslinked with deubiquitinases, E1 and E3s. In addition, compared to the classic Ub-VME and Ub-PA probes, Ub-AMA achieves improved labeling efficiency for E2 enzymes. Finally, proteomic profiling in cell lysates confirmed that Ub-AMA can capture E1, E2, and E3 enzymes as well as DUBs in a complex biological environment. In summary, the Ub-AMA probe developed here provides a new tool for in-depth analysis of the dynamic regulatory mechanisms of ubiquitination. Moreover, this work offers valuable insights for the design of other activity-based probes based on \u03b1,\u03b2-unsaturated carbonyl scaffolds.<\/jats:p>","DOI":"10.1021\/acschembio.6c00408","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T08:59:59Z","timestamp":1788253199000},"page":"2244-2252","source":"Crossref","is-referenced-by-count":0,"title":["Activity-Based Ubiquitin-\u03b1-amidomethyl Acrylate Probe for Profiling of Ubiquitinating Enzymes and Deubiquitinases"],"prefix":"10.1021","volume":"21","author":[{"given":"Luyu","family":"Shi","sequence":"first","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xu","family":"Li","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, China, 230026"]},{"name":"Qingdao University , , ,","place":["Qingdao, China, 266071"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingyue","family":"Zhang","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Han","family":"Wang","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, China, 230026"]},{"name":"Qingdao University , , ,","place":["Qingdao, China, 266071"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deguang","family":"Liu","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chuntong","family":"Li","sequence":"additional","affiliation":[{"name":"Zhengzhou University , , , ,","place":["Zhengzhou, Henan, China, 450001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0180-4265","authenticated-orcid":true,"given":"Jing","family":"Shi","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-5669-1509","authenticated-orcid":true,"given":"Lu-Jun","family":"Liang","sequence":"additional","affiliation":[{"name":"Zhengzhou University , , ,","place":["Zhengzhou, China, 450001"]},{"name":"University of Science and Technology of China , , ,","place":["Hefei, China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"key":"2026101511063709200_cit1","doi-asserted-by":"publisher","first-page":"203","DOI":"10.1146\/annurev-biochem-060310-170328","article-title":"The Ubiquitin Code","volume":"81","author":"Komander","year":"2012","journal-title":"Annu. Rev. Biochem."},{"key":"2026101511063709200_cit2","doi-asserted-by":"publisher","first-page":"399","DOI":"10.1038\/cr.2016.39","article-title":"Ubiquitin modifications","volume":"26","author":"Swatek","year":"2016","journal-title":"Cell Res."},{"key":"2026101511063709200_cit3","doi-asserted-by":"publisher","first-page":"273","DOI":"10.1038\/s41580-022-00543-1","article-title":"An expanded lexicon for the ubiquitin code","volume":"24","author":"Dikic","year":"2023","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"2026101511063709200_cit4","doi-asserted-by":"publisher","first-page":"503","DOI":"10.1146\/annurev.biochem.70.1.503","article-title":"Mechanisms Underlying Ubiquitination","volume":"70","author":"Pickart","year":"2001","journal-title":"Annu. Rev. Biochem."},{"key":"2026101511063709200_cit5","doi-asserted-by":"publisher","first-page":"626","DOI":"10.1038\/nrm.2016.91","article-title":"Structural insights into the catalysis and regulation of E3 ubiquitin ligases","volume":"17","author":"Buetow","year":"2016","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"2026101511063709200_cit6","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1146\/annurev-biochem-061516-044916","article-title":"Mechanisms of Deubiquitinase Specificity and Regulation","volume":"86","author":"Mevissen","year":"2017","journal-title":"Annu. Rev. Biochem."},{"key":"2026101511063709200_cit7","doi-asserted-by":"publisher","first-page":"338","DOI":"10.1038\/s41580-019-0099-1","article-title":"Breaking the chains: deubiquitylating enzyme specificity begets function","volume":"20","author":"Clague","year":"2019","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"2026101511063709200_cit8","doi-asserted-by":"publisher","first-page":"1242","DOI":"10.1038\/nm.3739","article-title":"Ubiquitination in disease pathogenesis and treatment","volume":"20","author":"Popovic","year":"2014","journal-title":"Nat. Med."},{"key":"2026101511063709200_cit9","doi-asserted-by":"publisher","first-page":"74","DOI":"10.1038\/ng0197-74","article-title":"De novo truncating mutations in E6-AP ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome","volume":"15","author":"Matsuura","year":"1997","journal-title":"Nat. Genet."},{"key":"2026101511063709200_cit10","doi-asserted-by":"publisher","first-page":"540","DOI":"10.1021\/ol403416w","article-title":"Dehydroalanine-Based Diubiquitin Activity Probes","volume":"16","author":"Haj-Yahya","year":"2014","journal-title":"Org. Lett."},{"key":"2026101511063709200_cit11","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202400659","article-title":"Chemical Tools for Probing the Ub\/Ubl Conjugation Cascades","volume":"26","author":"Kocha\u0144czyk","year":"2025","journal-title":"ChemBioChem"},{"key":"2026101511063709200_cit12","doi-asserted-by":"publisher","first-page":"12633","DOI":"10.1039\/D0SC03295F","article-title":"Development and application of ubiquitin-based chemical probes","volume":"11","author":"Sui","year":"2020","journal-title":"Chem. Sci."},{"key":"2026101511063709200_cit13","doi-asserted-by":"publisher","first-page":"1060","DOI":"10.1007\/s11426-024-1950-1","article-title":"Recent advances in chemical protein synthesis: method developments and biological applications","volume":"67","author":"Dong","year":"2024","journal-title":"Sci. China: Chem."},{"key":"2026101511063709200_cit14","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202203792","article-title":"Photocaging of Activity-Based Ubiquitin Probes via a C-Terminal Backbone Modification Strategy","volume":"61","author":"Wang","year":"2022","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026101511063709200_cit15","doi-asserted-by":"publisher","first-page":"1149","DOI":"10.1016\/S1074-5521(02)00248-X","article-title":"Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family","volume":"9","author":"Borodovsky","year":"2002","journal-title":"Chem. Biol."},{"key":"2026101511063709200_cit16","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202206205","article-title":"Expedient Synthesis of Ubiquitin-like Protein ISG15 Tools through Chemo-Enzymatic Ligation Catalyzed by a Viral Protease Lbpro","volume":"61","author":"Wang","year":"2022","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026101511063709200_cit17","doi-asserted-by":"publisher","first-page":"837","DOI":"10.1007\/s11426-022-1455-x","article-title":"Simultaneous capture of ISG15 conjugating and deconjugating enzymes using a semi-synthetic ISG15-Dha probe","volume":"66","author":"Li","year":"2023","journal-title":"Sci. China: Chem."},{"key":"2026101511063709200_cit18","doi-asserted-by":"publisher","first-page":"2873","DOI":"10.1021\/jacsau.3c00421","article-title":"Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells","volume":"3","author":"Liang","year":"2023","journal-title":"JACS Au"},{"key":"2026101511063709200_cit19","doi-asserted-by":"publisher","first-page":"2744","DOI":"10.1002\/anie.201611659","article-title":"Chemical Synthesis of Diubiquitin-Based Photoaffinity Probes for Selectively Profiling Ubiquitin-Binding Proteins","volume":"56","author":"Liang","year":"2017","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026101511063709200_cit20","doi-asserted-by":"publisher","first-page":"1442","DOI":"10.1021\/acscentsci.4c00642","article-title":"Chemical Synthesis of Human Proteoforms and Application in Biomedicine","volume":"10","author":"Ai","year":"2024","journal-title":"ACS Cent. Sci."},{"key":"2026101511063709200_cit21","doi-asserted-by":"publisher","first-page":"1155","DOI":"10.1016\/j.cclet.2018.03.022","article-title":"Biochemical properties of K11,48-branched ubiquitin chains","volume":"29","author":"Liang","year":"2018","journal-title":"Chin. Chem. Lett."},{"key":"2026101511063709200_cit22","doi-asserted-by":"publisher","first-page":"275","DOI":"10.1021\/cb9000348","article-title":"Ubiquitin C-Terminal Electrophiles Are Activity-Based Probes for Identification and Mechanistic Study of Ubiquitin Conjugating Machinery","volume":"4","author":"Love","year":"2009","journal-title":"ACS Chem. Biol."},{"key":"2026101511063709200_cit23","doi-asserted-by":"publisher","first-page":"2390","DOI":"10.1016\/j.chempr.2024.06.004","article-title":"Mirror-image protein and peptide drug discovery through mirror-image phage display","volume":"10","author":"Qi","year":"2024","journal-title":"Chem"},{"key":"2026101511063709200_cit24","doi-asserted-by":"publisher","first-page":"2515","DOI":"10.1007\/s11427-024-2860-5","article-title":"Advances in the chemical synthesis of human proteoforms","volume":"68","author":"Yang","year":"2025","journal-title":"Sci. China Life Sci."},{"key":"2026101511063709200_cit25","doi-asserted-by":"publisher","first-page":"1371","DOI":"10.1007\/s11426-019-9513-2","article-title":"Chimeric protein probes for C5a receptors through fusion of the anaphylatoxin C5a core region with a small-molecule antagonist","volume":"62","author":"Zuo","year":"2019","journal-title":"Sci. China: Chem."},{"key":"2026101511063709200_cit26","doi-asserted-by":"publisher","first-page":"17171","DOI":"10.1002\/anie.202105870","article-title":"Chemical Synthesis of Activity-Based E2-Ubiquitin Probes for the Structural Analysis of E3 Ligase-Catalyzed Transthiolation","volume":"60","author":"Liang","year":"2021","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026101511063709200_cit27","doi-asserted-by":"publisher","first-page":"972","DOI":"10.1038\/s41589-022-01067-7","article-title":"H2B Lys34 Ubiquitination Induces Nucleosome Distortion to Stimulate Dot1L Activity","volume":"18","author":"Ai","year":"2022","journal-title":"Nat. Chem. Biol."},{"key":"2026101511063709200_cit28","doi-asserted-by":"publisher","first-page":"668","DOI":"10.1038\/s41589-024-01750-x","article-title":"Mechanism of nucleosomal H2A K13\/15 monoubiquitination and adjacent dual monoubiquitination by RNF168","volume":"21","author":"Ai","year":"2025","journal-title":"Nat. Chem. Biol."},{"key":"2026101511063709200_cit29","doi-asserted-by":"publisher","first-page":"1221","DOI":"10.1016\/j.chempr.2023.01.012","article-title":"Synthetic E2-Ub-nucleosome conjugates for studying nucleosome ubiquitination","volume":"9","author":"Ai","year":"2023","journal-title":"Chem"},{"key":"2026101511063709200_cit30","doi-asserted-by":"publisher","first-page":"5187","DOI":"10.1093\/emboj\/20.18.5187","article-title":"A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14","volume":"20","author":"Borodovsky","year":"2001","journal-title":"EMBO J."},{"key":"2026101511063709200_cit31","doi-asserted-by":"publisher","first-page":"23878","DOI":"10.1021\/jacs.5c06399","article-title":"Design and Semisynthesis of Ubiquitin Extension Probes for Structural Analysis of Cullin1-Mediated Substrate Polyubiquitination","volume":"147","author":"Li","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"2026101511063709200_cit32","doi-asserted-by":"publisher","first-page":"334","DOI":"10.1038\/s41586-021-04097-8","article-title":"Structural insights into Ubr1-mediated N-degron polyubiquitination","volume":"600","author":"Pan","year":"2021","journal-title":"Nature"},{"key":"2026101511063709200_cit33","doi-asserted-by":"publisher","first-page":"412","DOI":"10.1007\/s11426-017-9189-6","article-title":"Semi-synthesis of disulfide-linked branched tri-ubiquitin mimics","volume":"61","author":"Si","year":"2018","journal-title":"Sci. China: Chem."},{"key":"2026101511063709200_cit34","doi-asserted-by":"publisher","first-page":"5871","DOI":"10.1007\/s11426-025-2697-6","article-title":"Synthesis of an E2-Ub-nucleosome conjugate to capture the E3 ligase PHF7-catalyzed H3K14 ubiquitination intermediate","volume":"68","author":"Tao","year":"2025","journal-title":"Sci. China: Chem."},{"key":"2026101511063709200_cit35","doi-asserted-by":"publisher","first-page":"239","DOI":"10.1038\/s41589-025-01985-2","article-title":"Structural basis for E4 enzyme Ufd2-catalyzed K48\/K29 branched ubiquitin chains","volume":"22","author":"Tong","year":"2025","journal-title":"Nat. Chem. Biol."},{"key":"2026101511063709200_cit36","doi-asserted-by":"publisher","first-page":"2148","DOI":"10.1021\/jacsau.5c00067","article-title":"Examining the Role of Threonine Phosphorylation in Ubiquitin\u2019s Function Using Chemical Protein Synthesis","volume":"5","author":"Wang","year":"2025","journal-title":"JACS Au"},{"key":"2026101511063709200_cit37","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmc.2024.118016","article-title":"RAD18-catalysed formation of ubiquitination intermediate mimic of proliferating cell nuclear antigen PCNA","volume":"117","author":"Zhang","year":"2025","journal-title":"Bioorg. Med. Chem."},{"key":"2026101511063709200_cit38","doi-asserted-by":"publisher","first-page":"476","DOI":"10.31635\/ccschem.019.20190001","article-title":"Chemical Synthesis of Structurally Defined Phosphorylated Ubiquitins Suggests Impaired Parkin Activation by Phosphorylated Ubiquitins with a Non-Phosphorylated Distal Unit","volume":"1","author":"Pan","year":"2019","journal-title":"CCS Chem."},{"key":"2026101511063709200_cit39","doi-asserted-by":"publisher","first-page":"4313","DOI":"10.1038\/s41467-025-59569-6","article-title":"Structural visualization of HECT-type E3 ligase Ufd4 accepting and transferring ubiquitin to form K29\/K48-branched polyubiquitination","volume":"16","author":"Wu","year":"2025","journal-title":"Nat. Commun."},{"key":"2026101511063709200_cit40","doi-asserted-by":"publisher","first-page":"523","DOI":"10.1038\/nchembio.2084","article-title":"A cascading activity-based probe sequentially targets E1\u2013E2\u2013E3 ubiquitin enzymes","volume":"12","author":"Mulder","year":"2016","journal-title":"Nat. Chem. Biol."},{"key":"2026101511063709200_cit41","doi-asserted-by":"publisher","first-page":"17817","DOI":"10.1021\/jacs.5c01732","article-title":"Ubiquitin Azapeptide Esters as Next-Generation Activity-Based Probes for Cysteine Enzymes in the Ubiquitin Signal Pathway","volume":"147","author":"Chanda","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"2026101511063709200_cit42","doi-asserted-by":"publisher","first-page":"7047","DOI":"10.1021\/jacs.0c00252","article-title":"Expressed Protein Ligation without Intein","volume":"142","author":"Qiao","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"2026101511063709200_cit43","doi-asserted-by":"publisher","first-page":"2483","DOI":"10.1038\/nprot.2013.152","article-title":"Chemical synthesis of proteins using peptide hydrazides as thioester surrogates","volume":"8","author":"Zheng","year":"2013","journal-title":"Nat. Protoc."},{"key":"2026101511063709200_cit44","doi-asserted-by":"publisher","first-page":"2031","DOI":"10.31635\/ccschem.023.202303397","article-title":"Ferricyanide-Promoted Oxidative Activation and Ligation of Protein Thioacids in Neutral Aqueous Media","volume":"6","author":"Chu","year":"2024","journal-title":"CCS Chem."},{"key":"2026101511063709200_cit45","doi-asserted-by":"publisher","first-page":"7645","DOI":"10.1002\/anie.201100996","article-title":"Protein Chemical Synthesis by Ligation of Peptide Hydrazides","volume":"50","author":"Fang","year":"2011","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026101511063709200_cit46","doi-asserted-by":"publisher","first-page":"10347","DOI":"10.1002\/anie.201203843","article-title":"Convergent Chemical Synthesis of Proteins by Ligation of Peptide Hydrazides","volume":"51","author":"Fang","year":"2012","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026101511063709200_cit47","doi-asserted-by":"publisher","first-page":"4135","DOI":"10.1021\/jacs.4c13464","article-title":"Mechanically Triggered Protein Desulfurization","volume":"147","author":"Han","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"2026101511063709200_cit48","doi-asserted-by":"publisher","first-page":"13333","DOI":"10.1002\/anie.201708067","article-title":"Practical Chemical Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-Linked Ub Isomer","volume":"56","author":"Tang","year":"2017","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026101511063709200_cit49","doi-asserted-by":"publisher","first-page":"2867","DOI":"10.1021\/ja309802n","article-title":"On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases","volume":"135","author":"Ekkebus","year":"2013","journal-title":"J. Am. Chem. Soc."}],"container-title":["ACS Chemical Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acschembio.6c00408","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/21\/9\/2244\/67415745\/cb-2026-00408p.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/21\/9\/2244\/67415745\/cb-2026-00408p.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T15:49:41Z","timestamp":1792079381000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acbcct\/article\/21\/9\/2244\/5329459\/Activity-Based-Ubiquitin-amidomethyl-Acrylate"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,26]]},"references-count":49,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,8,26]]},"published-print":{"date-parts":[[2026,9,18]]}},"URL":"https:\/\/doi.org\/10.1021\/acschembio.6c00408","relation":{},"ISSN":["1554-8929","1554-8937"],"issn-type":[{"value":"1554-8929","type":"print"},{"value":"1554-8937","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,26]]}}}