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 molecular glue interactomes. We map the targets of 20 molecular glues 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.靶向蛋白质降解（Targeted protein degradation）与诱导邻近（Induced proximity）均属于先进的分子干预策略，二者通过将靶蛋白募集至另一蛋白，以促进靶蛋白的修饰、调控或降解。随着分子胶（molecular glues）与降解剂（degraders）的生物学复杂性日益明晰，学界对无偏方法学的发展需求愈发迫切，以揭示未被发掘的化学靶点。本研究建立了一种基于细胞裂解液的高通量亲和纯化质谱（affinity purification mass spectrometry）工作流程，用于无偏鉴定分子胶相互作用组。本研究在两种正交细胞系中绘制了20种分子胶的靶点图谱，鉴定出270种被化学募集至CRBN的蛋白质，并证实了富集方法的优势：该方法可识别出全细胞全局筛选中常被忽略的新型蛋白靶点。本研究不仅大幅拓展了可靶向锌指转录因子的覆盖范围，更重要的是，鉴定出多种新型非锌指蛋白作为IMiD（immunomodulatory imide drugs）分子胶的靶点。本研究利用阿尔法折叠2（AlphaFold2）数据库获取的蛋白质结构进行序列比对，以此评估实验命中靶点的置信度，并通过实验验证了多个靶点，证实其直接相互作用及后续的泛素化修饰。本研究为CRBN的化学募集靶点提供了一份全面的清单，同时开发出一套稳健且可扩展的工作流程，用于在细胞裂解液中鉴定药物诱导的新型蛋白质相互作用。