Zhu Bing, Farris Tierra R, Milligan Sarah L, Chen Haosi, Zhu Ruijuan, Hong Aailing, Zhou Xiaochuan, Gao Xiaolian, McBride Jere W
Departments of Pathology University of Texas Medical Branch, Galveston, TX.
Departments of Microbiology & Immunology University of Texas Medical Branch, Galveston, TX.
Biochem Biophys Rep. 2016 Mar 1;5:430-438. doi: 10.1016/j.bbrep.2016.02.003.
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 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.
SUMO化和泛素化是真核细胞中参与重要生物过程调控的两种关键的翻译后修饰（PTM）。鉴定蛋白质中泛素（Ub）和小泛素相关修饰物（SUMO）共轭的赖氨酸残基对于理解泛素化和SUMO化的作用至关重要，但在实验上仍然具有挑战性。我们开发了一种强大的Ub/SUMO检测方法，该方法使用了一种新型的高密度肽阵列，该阵列集成在微流控装置中，可快速鉴定靶蛋白上的泛素化和SUMO化位点。我们用一组人类蛋白质和一种具有已知Ub或SUMO修饰靶位点的微生物效应物进行了检测，并确定这些蛋白质中有80%在先前使用传统方法确定的位点上被Ub或特定的SUMO异构体修饰。我们的结果在肽水平上证实了SUMO异构体和单个靶蛋白的特异性。总之，这种微流控高密度肽阵列方法是一种快速筛选检测方法，用于确定靶底物的Ub和SUMO修饰位点，这将为这些PTM靶位点的组成、选择性和特异性提供新的见解。
