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.