Comprehensive profiling of degrader-protein interactions

Targeted protein degradation and induced proximity both refer to advanced strategies that leverage the recruitment of target proteins to another protein to facilitate their modification, regulation or degradation. As the biological complexity of molecular glues and degraders has become increasingly apparent, there is a growing need for advances in unbiased methodology to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of degrader interactomes. We map the targets of 20 molecular glue degraders across two orthogonal cell lines identifying 270 proteins as chemically recruited to CRBN and demonstrate the power of enrichment methods for identifying novel protein targets that are often overlooked using global whole cell screening methods. We not only drastically increase the breadth of zinc finger transcription factors that are targetable, but more importantly identify many novel non-zinc finger proteins as targets of IMiD molecular glues. We use alignments with structures obtained from the Alphafold2 database as a method to estimate confidence in experimental hits and we experimentally validate several targets to show direct interaction and subsequent ubiquitylation. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.