Chip-Based Analysis of Protein-Protein Interactions by Fluorescence Detection and On-Chip Immunoprecipitation Combined with LC-MS/MS A
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- 作者：Jens R. Sydor ; Mark Scalf ; Steve Sideris ; Guo Dong Mao ; Yash Pandey ; Ming Tan ; Maria Mariano ; Michael F. Moran ; Steffen Nock ; and Peter Wagner
- 刊名：Analytical Chemistry
- 出版年：2003
- 出版时间：November 15, 2003
- 年：2003
- 卷：75
- 期：22
- 页码：6163 - 6170
- 全文大小：237K
- 年卷期：v.75,no.22(November 15, 2003)
- ISSN：1520-6882
文摘
A new chip-based method to identify protein-proteininteractions was developed using the guanine nucleotideexchange factor GRF2 and two interacting proteins, Rasand calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented bypresentation of an oriented anti-FLAG antibody. A flowcell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with thetwo analytes was verified by fluorescence assays. On-chiptryptic digest assays were then performed on the capturesurface and analyzed by 
LC-MS/MS. The interaction ofGRF2 with calmodulin and Ras was demonstrated, andthe lower limit of detection was determined. We alsoimplemented an on-chip immunoprecipitation assay toidentify GRF2-binding partners from complex proteinmixtures. Cells overexpressing FLAG-GRF2 were lysedand then incubated with the anti-FLAG chip. In additionto detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identifyendogenous levels of proteins, bound to recombinant baitproteins. This chip-based method has the advantage thatno subsequent gel separations of protein complexes priorto LC-MS analysis are required and is therefore amenableto miniaturized high-throughput determination of protein-protein interactions.
LC-MS/MS. The interaction ofGRF2 with calmodulin and Ras was demonstrated, andthe lower limit of detection was determined. We alsoimplemented an on-chip immunoprecipitation assay toidentify GRF2-binding partners from complex proteinmixtures. Cells overexpressing FLAG-GRF2 were lysedand then incubated with the anti-FLAG chip. In additionto detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identifyendogenous levels of proteins, bound to recombinant baitproteins. This chip-based method has the advantage thatno subsequent gel separations of protein complexes priorto LC-MS analysis are required and is therefore amenableto miniaturized high-throughput determination of protein-protein interactions.