{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T17:51:32Z","timestamp":1791222692394,"version":"4.1.0"},"posted":{"date-parts":[[2026,9,30]]},"group-title":"Biochemistry","reference-count":51,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T00:00:00Z","timestamp":1790726400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"National Institutes of Health","award":["RM1GM149374"],"award-info":[{"award-number":["RM1GM149374"]}]},{"name":"National Institutes of Health","award":["GM144293"],"award-info":[{"award-number":["GM144293"]}]},{"name":"Department of Education","award":["P200A240072"],"award-info":[{"award-number":["P200A240072"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,30]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Proteolysis-targeting chimeras (PROTACs) promote targeted protein degradation by inducing a transient ternary complex between a protein of interest and an E3 ubiquitin ligase. Despite the clinical promise of these event-driven therapeutics, defining the conformational landscape and interfacial organization of these complexes remains challenging. Here, we use an integrated native mass spectrometry (nMS) approach combining ion mobility, surface-induced dissociation (SID), and variable-temperature electrospray ionization (vT-ESI) to determine how small-molecule architecture shapes the structural and thermodynamic landscapes of bromodomain-containing protein 4 (BRD4) with the VCB E3 ligase complex. Using a set of PROTACs that differ in targeting ligand identity (JQ1 versus I-BET726) and PEG linker length, ion mobility measurements show that shorter linkers constrain the ternary complex into more extended topologies, whereas longer, more flexible linkers allow dynamic sampling of relaxed, native-like conformations. SID directly probes noncovalent interfaces and shows that JQ1 complexes favor prompt PROTAC ejection, whereas tighter-binding I-BET726 complexes are more likely to retain the PROTAC at the interface. Complementing these kinetically-controlled gas-phase measurements, vT-ESI reveals a monophasic melting transition for a JQ1-mediated complex and a distinct biphasic melting profile for an I-BET726-mediated complex. The gas-phase activation measurements revealed targeting-ligand-driven differences in PROTAC ejection and retention, whereas vT-ESI uncovered distinct temperature-dependent disassembly pathways for the matched-linker-length JQ10L and IBET10L complexes. Together, these complementary techniques show how PROTAC architecture influences ternary-complex conformation, interfacial connectivity, and disassembly.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Synopsis<\/jats:title>\n                  <jats:p>Proteolysis-targeting chimeras (PROTACs) offer a promising therapeutic strategy by selectively directing disease-related proteins for intracellular degradation. Here, we employ a systematic native mass spectrometry approach to investigate how PROTAC design impacts the conformation and stability of the resulting ternary complexes. Integrating gas- and solution-phase data provides critical insights to inform rational PROTAC optimization.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.29.755516","type":"posted-content","created":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T17:50:30Z","timestamp":1790790630000},"source":"Crossref","is-referenced-by-count":0,"title":["Native Mass Spectrometry Reveals Distinct Effects of Linker Length and Targeting-Ligand Identity on PROTAC Ternary Complexes"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-1120-0889","authenticated-orcid":false,"given":"Evan N.","family":"Whitford","sequence":"first","affiliation":[{"name":"School of Chemistry and Biochemistry and Native Mass Spectrometry Guided Structural Biology Center, Georgia Institute of Technology, Atlanta, GA 30332, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua D.","family":"Gilbert","sequence":"additional","affiliation":[{"name":"Native Mass Spectrometry Guided Structural Biology Center, The Ohio State University, Columbus, OH, 43210, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marius M.","family":"Kostelic","sequence":"additional","affiliation":[{"name":"Native Mass Spectrometry Guided Structural Biology Center, The Ohio State University, Columbus, OH, 43210, United States"},{"name":"Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, 43210, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle A.","family":"Estrada","sequence":"additional","affiliation":[{"name":"Technology and Therapeutic Platforms, AbbVie, 1 N. Waukegan Rd., North Chicago, IL, 60064, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron C.","family":"Ehlinger","sequence":"additional","affiliation":[{"name":"Target Enabling Technologies, AbbVie, 1 N. Waukegan Rd., North Chicago, IL, 60064, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiffany A.","family":"Thibaudeau","sequence":"additional","affiliation":[{"name":"Target Enabling Technologies, AbbVie, 1 N. Waukegan Rd., North Chicago, IL, 60064, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaun M.","family":"McLoughlin","sequence":"additional","affiliation":[{"name":"Technology and Therapeutic Platforms, AbbVie, 1 N. 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