{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,9]],"date-time":"2025-11-09T17:53:01Z","timestamp":1762710781522,"version":"build-2065373602"},"reference-count":28,"publisher":"Wiley","issue":"14","license":[{"start":{"date-parts":[[2016,7,21]],"date-time":"2016-07-21T00:00:00Z","timestamp":1469059200000},"content-version":"vor","delay-in-days":20,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100000837","name":"University of Nottingham","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000837","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteomics"],"published-print":{"date-parts":[[2016,7]]},"abstract":"<jats:p>Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate\u2010conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker\u2010optimized ubiquitin\u2010binding domain hybrid (t\u2010UBD) containing two UBDs, a ZnF\u2010UBP domain in tandem with a linkage\u2010selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48\u2010linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48\u2010linked ubiquitin dimers. Furthermore, MS\/MS analyses indicate that the t\u2010UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48\u2010linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain\u2010selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the \u2018ubiquitome\u2019. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (<jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/proteomecentral.proteomexchange.org\/dataset\/PXD004059\">http:\/\/proteomecentral.proteomexchange.org\/dataset\/PXD004059<\/jats:ext-link>).\n<\/jats:p>","DOI":"10.1002\/pmic.201600067","type":"journal-article","created":{"date-parts":[[2016,4,2]],"date-time":"2016-04-02T13:38:48Z","timestamp":1459604328000},"page":"1961-1969","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Mass spectrometry insights into a tandem ubiquitin\u2010binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin"],"prefix":"10.1002","volume":"16","author":[{"given":"Daniel","family":"Scott","sequence":"first","affiliation":[{"name":"School of Life Sciences Queen's Medical Centre University of Nottingham  Nottingham UK"},{"name":"School of Chemistry University Park University of Nottingham  Nottingham UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tom P","family":"Garner","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Albert Einstein College of Medicine of Yeshiva University  Bronx NY USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jed","family":"Long","sequence":"additional","affiliation":[{"name":"School of Chemistry University Park University of Nottingham  Nottingham UK"},{"name":"Centre for Biomolecular Sciences University Park University of Nottingham  Nottingham UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jo","family":"Strachan","sequence":"additional","affiliation":[{"name":"Institute of Cell Biology University of Edinburgh  Edinburgh UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sharad C.","family":"Mistry","sequence":"additional","affiliation":[{"name":"Protein &amp; Nucleic Acid Chemistry Laboratory University of Leicester  Leicester UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrew R.","family":"Bottrill","sequence":"additional","affiliation":[{"name":"Protein &amp; Nucleic Acid Chemistry Laboratory University of Leicester  Leicester UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David J.","family":"Tooth","sequence":"additional","affiliation":[{"name":"School of Life Sciences Queen's Medical Centre University of Nottingham  Nottingham UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark S.","family":"Searle","sequence":"additional","affiliation":[{"name":"School of Chemistry University Park University of Nottingham  Nottingham UK"},{"name":"Centre for Biomolecular Sciences University Park University of Nottingham  Nottingham UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Neil J.","family":"Oldham","sequence":"additional","affiliation":[{"name":"School of Chemistry University Park University of Nottingham  Nottingham UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rob","family":"Layfield","sequence":"additional","affiliation":[{"name":"School of Life Sciences Queen's Medical Centre University of Nottingham  Nottingham UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2016,7,21]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1042\/BST0370937"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-060310-170328"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature08247"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2010.03.029"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature09976"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.273730"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2013.08.016"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-051810-094654"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi201137e"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2006.02.038"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2005.05.013"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2015.01.042"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2009.02.011"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M800947200"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1021\/pr201167n"},{"key":"e_1_2_8_17_1","first-page":"1","article-title":"Broad utility of an affinity\u2010enrichment strategy for unanchored polyubiquitin chains","volume":"7","author":"Strachan J.","year":"2013","journal-title":"J. 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