Proteome-wide target engagement and ternary complex mapping of molecular glues using LiP-MS

Deciphering molecular targets and interaction networks of small molecules within native cellular contexts remains essential for both target-based and phenotypic drug discovery. This is particularly critical for molecular glues (MGs), which promote selective protein degradation through induced protein-protein interactions. Two mechanistic axes define MG action: (1) direct binding of the compound to an E3 ligase that subsequently recruits a neosubstrate, and (2) compound engagement with a primary protein target that promotes E3 ligase recruitment and ternary complex formation. Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful, label-free approach for elucidating small-molecule target engagement and mapping binding sites in complex proteomes without chemical tagging or genetic manipulation. Here, we expand the application of LiP-MS to two complementary experimental formats that interrogate distinct stages of molecular glue activity. In the first scenario, an in-lysate LiP-MS workflow was implemented to identify primary drug-protein interactions across the proteome. Using quantitative data-independent acquisition mass spectrometry (DIA-MS) and a seven-point concentration series, we monitored conformational and accessibility changes across >250,000 peptides from >8,000 proteins. Machine learning-based LiP scoring enabled peptide-level resolution of binding sites and quantitative ranking of target engagement. In the second scenario, a live-cell LiP-MS assay was developed to capture compound-induced protein-protein interaction changes under physiological conditions. This live-cell format enables detection of secondary, compound-dependent protein recruitment events, including ternary complex formation with E3 ligases and other associated proteins. To evaluate LiP-MS performance in characterizing molecular glue mechanisms, we conducted global target identification experiments using two representative compounds: SR-4835, a cyclin K degrader that binds CDK12 and recruits DDB1, and MRT-2359, a GSPT1 degrader that engages CRBN. Live-cell LiP-MS experiments were performed at two time points (1 hour and 6 hours) to monitor compound-dependent conformational changes and protein recruitment dynamics associated with primary target engagement and potential ternary complex formation. Together, LiP-MS provides a comprehensive, high-resolution platform for mapping small-molecule target engagement and for characterizing dynamic protein recruitment events associated with molecular glue activity directly in the cellular environment.