Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array

• A high throughput in vitro Ub/SUMO assay using microfluidic high density peptide array was developed.
• The sensitivity and specificity of the assay was validated using human and bacterial proteins with known Ub/SUMO sites.
• Specificity for SUMO isoforms on individual target proteins was determined at the peptide level.
• This approach can rapidly identify specific Ub/SUMO sites in unknown substrates.

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.