Führer Siska, Gallant Kai, Kaschani Farnusch, Kaiser Markus, Janning Petra, Waldmann Herbert, Gersch Malte
Chemical Genomics Centre, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 15, 44227, Dortmund, Germany.
Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227, Dortmund, Germany.
Angew Chem Int Ed Engl. 2025 Aug 4;64(32):e202508916. doi: 10.1002/anie.202508916. Epub 2025 Jun 26.
Small molecules that alter protein ubiquitination are emerging as therapeutics due to their ability to modulate targets previously deemed undruggable. These compounds comprise PROTACs, molecular glue degraders, and DUB inhibitors, among others. However, methods for the proteome-wide monitoring of compound-induced changes in protein polyubiquitination, which may also detect non-degradative modifications, are lacking. Here, we report the utilization of polyubiquitin enrichment coupled to mass spectrometry to monitor small molecule-induced changes in cellular protein ubiquitination. We established enrichment through tandem ubiquitin binding entities (TUBEs) following semi-denaturing cell lysis and devised an elution protocol compatible with downstream LC-MS/MS analysis. We demonstrate the broad applicability of the workflow by assessing ubiquitination changes induced by a PROTAC, a p97 inhibitor, and deubiquitinase inhibitors. Application of the assay to compounds inhibiting the deubiquitinase USP7 revealed the induction of non-degradative ubiquitination on the E3 ligase UBE3A. Collectively, we established a versatile proteomics method to facilitate the direct investigation of cellular polyubiquitination, with high relevance for the identification and characterization of protein degraders, stabilizers, and other molecules with ubiquitin-mediated bioactivity.
由于能够调节先前被认为不可成药的靶点，改变蛋白质泛素化的小分子正逐渐成为治疗药物。这些化合物包括PROTAC、分子胶降解剂和去泛素化酶抑制剂等。然而，缺乏在蛋白质组范围内监测化合物诱导的蛋白质多聚泛素化变化的方法，而这种变化也可能检测到非降解性修饰。在这里，我们报告了利用多聚泛素富集结合质谱来监测小分子诱导的细胞蛋白质泛素化变化。我们在半变性细胞裂解后通过串联泛素结合实体（TUBE）建立了富集，并设计了一种与下游液相色谱-串联质谱（LC-MS/MS）分析兼容的洗脱方案。我们通过评估PROTAC、p97抑制剂和去泛素化酶抑制剂诱导的泛素化变化，证明了该工作流程的广泛适用性。将该检测方法应用于抑制去泛素化酶USP7的化合物，揭示了E3泛素连接酶UBE3A上非降解性泛素化的诱导。总的来说，我们建立了一种通用的蛋白质组学方法，以促进对细胞多聚泛素化的直接研究，这对于鉴定和表征蛋白质降解剂、稳定剂以及其他具有泛素介导生物活性的分子具有高度相关性。
