Chip-based analysis of SUMO (small ubiquitin-like modifier) conjugation to a target protein

Abstract

A chip-based analysis of protein interactions and modifications in cell signaling pathways has been of great potential in drug discovery, diagnostics, and cell biology, because it enables rapid and high-throughput biological assays with a small amount of samples. We report a chip-based analysis of sumoylation, the post-translational modification (PTM) process that involves covalent attachment of the small ubiquitin-like modifier (SUMO) protein to a target protein through multiple enzyme reactions in eukaryofic cells. Substrate proteins were spotted onto a glass surface followed by the addition of the reaction mixture for surnoylation, and the SUMO conjugation was readily detected by using fluorescent dye-labeled antibody. Under the optimized condition, on-chip surnoylation of Ran GTPase-activating protein 1(RanGAP1) domain resulted in highly specific fluorescence intensity compared to that of its mutant (K524A) irrelevant to SUMO conjugation. The on-chip sumoylation was also verified and quantified by using the surface plasmon resonance(SPR) spectroscopy. As the exemplary study for a parallel analysis of surnoylation, fluorescent detection of surnoylation was conducted in a microarray format on a glass slide. The chip-based analysis developed here is expected to be applicable to assay for screening of target proteins from existing protein pools and proteome arrays in a high throughput manner. (c) 2006 Elsevier B.V. All rights reserved.