Targeted protein degradation targeting conventionally undruggable proteins is a transformative approach in drug discovery. Molecular glues (MGs) achieve this by enhancing weak intrinsic interactions between targets and E3 ligase, enabling ubiquitin-proteasome-mediated degradation. While online affinity selection mass spectrometry (MS) struggles to identify weak binders due to on-column dissociation and lack of ternary complex interrogation, native MS facilitates the direct identification of E3-MG-target complexes. However, manual sample preparation and direct infusion limit its throughput.
To address throughput limitation and on-column dissociation, we multiplexed four compounds per well and employed a 3-cm prototype SEC column for rapid online buffer exchange. Native MS (nMS) screening of all 96 compounds was completed within an hour, achieving a throughput exceeding 2,500 compounds per day. Strong ternary complex formation between CRBN–DDB1 and WEE1 was detected in four mixtures, with two additional samples showing moderate binding. Identifying individual binders within multiplexed samples was complicated by native adduct interference and nonspecific interactions. To resolve this, ternary complexes were isolated in the quadrupole and subjected to collision-induced dissociation. Released ligands, potentially uncharged in the gas phase, were detected via polarity switching. MS2 enabled accurate mass determination and comparison with the compound library, while MS3 provided fragmentation-based structural elucidation of unknowns. This integrated MS1–MS3 workflow enhanced throughput and specificity, and subsequent individual screening confirmed 16 of 96 compounds as potential molecular glues with varying binding affinities.
This nMS-based workflow enhances screening throughput, resolves compound ambiguity, and facilitates discovery of novel MGs. It enables multiplex screening and analysis of over 2,500 compounds per day. Selected hits were further characterized using cryo-EM, yielding high-resolution (2.7 Å) structures of WEE1-MG-CRBN-DDB1 ternary complexes reflecting the drug binding pocket. These structures reveal how MGs mediate and stabilize protein–protein interactions, offering critical mechanistic insights to guide drug design and optimization.