Chip-based analysis of SUMO (small ubiquitin-like modifier) conjugation to a target protein.
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 eukaryotic cells. Substrate proteins were spotted onto a glass surface followed by the addition of the reaction mixture for sumoylation, and the SUMO conjugation was readily detected by using fluorescent dye-labeled antibody. Under the optimized condition, on-chip sumoylation 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 sumoylation, fluorescent detection of sumoylation 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.
Oh
Young-Hee
YH
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Daejon 305-701, Republic of Korea.
Hong
Mi-Young
MY
Jin
Zongwen
Z
Lee
Taeryong
T
Han
Min-Kyu
MK
Park
Sunyoung
S
Kim
Hak-Sung
HS
D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational; D025842:SUMO-1 Protein / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism
D020690:GTPase-Activating Proteins; C092317:RANGAP1 protein, human; D025842:SUMO-1 Protein