title: Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.
abstract: Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection.
authors.affiliation: Department of Neuroscience and High Throughput Biology Center, School of Medicine, Johns Hopkins University, BRB311, 733 North Broadway, Baltimore, MD 21205, USA.
authors.forename: Meng
authors.initials: M
authors.lastname: Wu
authors.forename: Brian
authors.initials: B
authors.lastname: Coblitz
authors.forename: Sojin
authors.initials: S
authors.lastname: Shikano
authors.forename: Shunyou
authors.initials: S
authors.lastname: Long
authors.forename: Matt
authors.initials: M
authors.lastname: Spieker
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Frutos
authors.forename: Sunil
authors.initials: S
authors.lastname: Mukhopadhyay
authors.forename: Min
authors.initials: M
authors.lastname: Li
mesh_terms: D048948:14-3-3 Proteins / Q000276:immunology* / Q000378:metabolism*; D045163:Antibodies, Phospho-Specific / Q000276:immunology*; D015374:Biosensing Techniques / Q000379:methods*; D015394:Molecular Structure; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D012680:Sensitivity and Specificity
chemical_list: D048948:14-3-3 Proteins; D045163:Antibodies, Phospho-Specific; D010748:Phosphopeptides