Chip-Based Analysis of Protein−Protein Interactions by Fluorescence Detection and On-Chip Immunoprecipitation Combined with μLC−MS/MS Analysis

• Published in: Analytical chemistry (Washington) 2003-11, Vol.75 (22), p.6163-6170
• Main Authors: Sydor, Jens R, Scalf, Mark, Sideris, Steve, Mao, Guo Dong, Pandey, Yash, Tan, Ming, Mariano, Maria, Moran, Michael F, Nock, Steffen, Wagner, Peter
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Analytical, structural and metabolic biochemistry; Antibodies; Biological and medical sciences; Calcium-binding protein; Calmodulin; Chemistry; Chromatographic methods and physical methods associated with chromatography; Exact sciences and technology; Fluorescence; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; guanine nucleotide exchange factor; Immobilization; Immunoprecipitation; Other chromatographic methods; Protein interaction; Proteins; Q1; Ras protein; Spectrometric and optical methods; streptavidin
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac034258u

A new chip-based method to identify protein−protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by μLC−MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC−MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein−protein interactions.