Native Mass Spectrometry Reveals Distinct Effects of Linker Length and Targeting-Ligand Identity on PROTAC Ternary Complexes
- 1Georgia Institute of Technology
Abstract
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.
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BibTeX
@misc{whitford2026native,
title = {{Native Mass Spectrometry Reveals Distinct Effects of Linker Length and Targeting-Ligand Identity on PROTAC Ternary Complexes}},
author = {E. N. Whitford and J. D. Gilbert and M. M. Kostelic and M. A. Estrada and A. C. Ehlinger and T. A. Thibaudeau and S. M. McLoughlin and V. H. Wysocki},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.29.755516},
url = {https://www.biorxiv.org/content/10.64898/2026.09.29.755516v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Native Mass Spectrometry Reveals Distinct Effects of Linker Length and Targeting-Ligand Identity on PROTAC Ternary Complexes
AU - E. N. Whitford
AU - J. D. Gilbert
AU - M. M. Kostelic
AU - M. A. Estrada
AU - A. C. Ehlinger
AU - T. A. Thibaudeau
AU - S. M. McLoughlin
AU - V. H. Wysocki
PY - 2026
DA - 2026/09/29
PB - bioRxiv
DO - 10.64898/2026.09.29.755516
UR - https://www.biorxiv.org/content/10.64898/2026.09.29.755516v1
AB - 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.
ER -