Selective lysine ubiquitination using activated phenol esters

Abstract

Ubiquitination of target proteins is an essential post-translational modification influencing a wide variety of cellular processes. We here describe the use of a novel water-soluble acylation reagent based on the 2,4-dichloro-6-sulfonic acid phenol ester of ubiquitin for efficient and selective ubiquitin modification of peptides. Under alkaline conditions this reagent is swift and regioselective towards lysine acylation, while at neutral pH it shows loss of regioselectivity and is able to acylate both lysine- and N-terminal modification at reduced speeds. As a proof of concept we utilize a model peptide to demonstrate this strategy proving to be successful. We then proceeded with the ubiquitination of a synthetic protein called FUBI under alkaline conditions followed by tandem MS analysis, proving the selective lysine ubiquitination works to prepare protein-protein conjugates.