Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.
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.
Wu
Meng
M
Department of Neuroscience and High Throughput Biology Center, School of Medicine, Johns Hopkins University, BRB311, 733 North Broadway, Baltimore, MD 21205, USA.
Coblitz
Brian
B
Shikano
Sojin
S
Long
Shunyou
S
Spieker
Matt
M
Frutos
Anthony G
AG
Mukhopadhyay
Sunil
S
Li
Min
M
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
D048948:14-3-3 Proteins; D045163:Antibodies, Phospho-Specific; D010748:Phosphopeptides