Mass spectrometric analysis of small-molecule-induced ubiquitination and deubiquitination

Abstract

The therapeutic targeting of the ubiquitin system is a growing field in chemical biology. Thus, the need to reliably detect small-molecule-induced alterations of protein ubiquitination is emerging. At the same time, there is a lack of methods for the unbiased and broad screening of small molecules for their ability to target the ubiquitin system. This thesis describes the establishment of a mass-spectrometric method to detect changes in the cellular polyubiquitome upon treatment of mammalian cells with different small molecules. The assay involves the enrichment of polyubiquitinated proteins from cell lysate using a tandem ubiquitin binding entity, followed by the digestion of the polyubiquitome, and its analysis by LC-MS/MS. Application to different small molecule treatments showed the enrichment of PROTAC target proteins from cells with inhibited proteasome. The method further demonstrated its versatility in the context of an intact proteasome and deubiquitinase inhibitors by also covering non-degradative ubiquitination. Finally, applying the method to uncharacterized research compounds showed the versatility and quality of the assay to recognize subtle changes in protein polyubiquitination. Collectively, the enrichment of polyubiquitinated proteins coupled to mass spectrometry provides a valuable addition to the repertoire of proteomics-based methods for the discovery and characterization of small molecules targeting the ubiquitin system.