Abstract
Ubiquitin (Ub) proteoforms control nearly every aspect of eukaryotic cell biology through their diversity. Inspired by the widely used Ub C-terminal electrophiles (Ub-E), here we report the identification of multivalent binding of Ub with deubiquitylating enzymes (Dubs) using genetic code expansion (GCE) and crosslinking mass spectrometry. While the Ub-Es only gather structural information with the S1 Dub sites, we demonstrate that GCE of Ub with p-benzoyl-L-phenylalanine enables identification of interaction modes beyond the S1 site with a panel of Dubs of both eukaryotic and prokaryotic origin. Collectively, this represents the next generation of Ub-based affinity probes with a unique ability to unravel Ub interaction landscapes beyond what is afforded by cysteine-based chemistries.