LaBaer Lab | Technology
Revolutionizing high throughput protein interaction studies
Investigators: Sanjeeva Srivastava, Ph.D., Manuel Fuentes, Ph.D.
Collaborator: Dale Larson (Draper Labs)
In the post-genome era having sequenced the human genome, one of the most important pursuits is to understand the function of proteins. The study of biochemical activity of gene products has led to better understanding of many cellular signaling pathways and their regulation. These pathways have served as a foundation to develop models for the pathogenesis of various diseases, including cancer. This in turn has led to the development of various diagnostics and improved therapeutics. In order to increase the speed with which we explore the molecular pathways of disease, we have embraced various genomics and proteomics approaches to study biology at large scale. Thus far, this effort has generated immense collections of data which enhanced our biochemical understanding of protein function. However, most current proteome-scale technologies are fairly crude end point assays that provide little quantitative information compared with standard biochemical approaches. In this project, we are coupling two technologies (NAPPA and SPRi) that will result in a high-throughput platform to detect and characterize a variety of protein interactions using a label-free detection system that is sensitive, quantitative and provides information on binding kinetics. We have developed new chemistries for NAPPA (based on interaction of E-coil/K-coil, and K-coil as capture reagent) compatible with a surface plasmon resonance imaging (SPRi) device that has been adapted to be compatible with protein microarrays and detecting multiplex binding events. SPR is based upon the creation of surface plasmons, oscillations of free electrons that propagate parallel to a metal/dielectric interface.
The method measures changes in refractive index very close to a sensor surface which are dependent on the dielectric properties of the metal, which in turn depend on what proteins are present on the surface. The method allows for real time and label-free detection of binding events. We have used NAPPA to express proteins on the array at the time of assay and using a label-free approach we have measured binding events (Figure 1). This enabled the direct and rapid determination of association and dissociation rates of binding, determination of strength of the binding and specificity of interactions at large scale. We have now made a collection of more than 60 known clinical mutations in the p53 protein and have measured the consequences of these mutations to MDM2 binding in a single experiment. This technology has potential to revolutionize the study of protein interaction networks by enabling quantitative comparisons of binding affinities across many molecular species, as well as determining the kinetic data of interactions pathways.
SPRi detection of proteins expressed by NAPPA. The indicated proteins were expressed on the array and specific antibodies added. Antibody binding is indicated by deflection of the sensogram. These curves enable computation of kon and koff.