title: Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array.
abstract: 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 powerfulin vitroUb/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.
authors.affiliation: Departments of Pathology University of Texas Medical Branch, Galveston, TX.
authors.forename: Bing
authors.initials: B
authors.lastname: Zhu
authors.affiliation: Departments of Microbiology & Immunology University of Texas Medical Branch, Galveston, TX.
authors.forename: Tierra R
authors.initials: TR
authors.lastname: Farris
authors.affiliation: Departments of Pathology University of Texas Medical Branch, Galveston, TX.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Milligan
authors.affiliation: Department of Biology and Biochemistry, University of Houston, TX.
authors.forename: Haosi
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Biology and Biochemistry, University of Houston, TX.
authors.forename: Ruijuan
authors.initials: R
authors.lastname: Zhu
authors.affiliation: LC Sciences, Houston, TX.
authors.forename: Aailing
authors.initials: A
authors.lastname: Hong
authors.affiliation: LC Sciences, Houston, TX.
authors.forename: Xiaochuan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Biology and Biochemistry, University of Houston, TX.
authors.forename: Xiaolian
authors.initials: X
authors.lastname: Gao
authors.affiliation: Departments of Pathology University of Texas Medical Branch, Galveston, TX; Departments of Microbiology & Immunology University of Texas Medical Branch, Galveston, TX; Center for Biodefense and Emerging Infectious Diseases University of Texas Medical Branch, Galveston, TX; Sealy Center for Vaccine Development University of Texas Medical Branch, Galveston, TX; Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX.
authors.forename: Jere W
authors.initials: JW
authors.lastname: McBride
keywords: Ehrlichia; SUMO; effector; high density peptide array; ubiquitin