TY - JOUR
T1 - Small Molecule-Induced Alterations of Protein Polyubiquitination Revealed by Mass-Spectrometric Ubiquitome Analysis
AU - Führer, Siska
AU - Gallant, Kai
AU - Kaschani, Farnusch
AU - Kaiser, Markus
AU - Janning, Petra
AU - Waldmann, Herbert
AU - Gersch, Malte
N1 - Publisher Copyright:
© 2025 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.
PY - 2025/8/4
Y1 - 2025/8/4
N2 - 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.
AB - 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.
KW - Biological activity
KW - Drug discovery
KW - Protein modifications
KW - Proteomics
KW - Ubiquitin proteasome system
UR - https://www.scopus.com/pages/publications/105009307676
U2 - 10.1002/anie.202508916
DO - 10.1002/anie.202508916
M3 - Article
C2 - 40444580
AN - SCOPUS:105009307676
SN - 1433-7851
VL - 64
JO - Angewandte Chemie - International Edition
JF - Angewandte Chemie - International Edition
IS - 32
M1 - e202508916
ER -