E3 and DUB substrate identification, and chain linkage profiling using microarrays

E3 ubiquitin ligases are the largest family of proteins in humans (~620), and mostly determine the substrate specificity of ubiquitin transfer to target proteins. De‐ubiquitylase enzymes (DUBs), in contrast, remove ubiquitin from proteins to recycle ubiquitin, to modify the nature of the linkage (editing), to terminate the signal, or to salvage protein from degradation. In between these two enzyme families is the ubiquitin itself, which is found singly on proteins as well as in chains of various length and linkage, i.e., a reference to which lysines are used to join adjacent moieties. Here we report using E1, E2, and E3 enzymes with free ubiquitin and ATP to transfer ubiquitin to immobilized proteins on microarrays. E3's were first screened against a nearly comprehensive panel of human E2's to determine optimal E2/E3 pairings. Here we show that various E3's ubiquitylate distinct but overlapping sets of protein substrates, which were also confirmed in vitro. We also demonstrate serial enzymatic activity on the microarray in which the protein substrates are first ubiquitylated, washed free of ubiquitylation machinery, then treated with DUB enzymes to profile their substrate specificities. These experiments were performed with wild‐type (untagged) ubiquitin as well as single‐lysine mutants to characterize the linkage specificities of the enzymes. Finally, we were able to differentiate monoubiquitylation and polyubiquitylation of substrates by comparing identically treated arrays visualized with Tandem Ubiquitin Binding Entities (TUBEs) which are specific to polyubiquitin, and antibody capable of recognizing both mono and poly‐ubiquitin. We believe the microarray format will prove critical to rapidly elucidating the substrates of E3 and DUB enzymes, as well as elucidating the chain preferences of both.