Ubiquitination site analysis of eGFP by DARPin-based bioPROTACs analysed by LC-MS-MS

We investigated design principles of DARPin-based bioPROTACs targeting the model substrate GS-eGFP (eGFP with an n-terminal tag, necessary for proteasomal unfolding). Using a highly active E3 ligase domain from CHIP and nine well-characterized DARPins, we evaluated their ability to induce proteasomal degradation. Seven of nine DARPin bioPROTACs successfully mediated catalytic degradation of GS-eGFP, with the binding epitope emerging as a key determinant of efficiency. LC-MS/MS analysis revealed that efficient degradation required ubiquitination at four specific lysine residues on eGFP. Notably, DP6-bioPROTAC promoted polyubiquitination, whereas DP9-bioPROTAC did not, likely due to differences in binding geometry. DP7-bioPROTAC appeared to hinder E2 enzyme access, further confirming the importance of structural orientation. Additionally, we observed that DARPins lacking an E3 domain can trigger non-catalytic degradation, potentially via recruitment of endogenous E3 ligases. Ubiquitination patterns of selected GS-eGFP complexes (DP1, DP9, and a lysine-free DP6 mutant) were also characterized by LC-MS/MS.