STTR Phase I: Single molecule sequencing of phosphorylated proteins for next-generation protein analyses and diagnostics

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) Phase I project will be to develop a highly sensitive assay that characterizes protein modifications for life science research applications. Proteins are life's nanomachines and serve as the targets for almost all drugs and the vast majority of diagnostic tests. One type of Modifications to proteins, such as addition of phosphate molecules (termed phosphorylation), are key triggers that alter protein activity. Subsequently, this can radically change cellular behaviour, such as affecting embryonic growth or development of tumors. The market for technologies studying proteins and their modifications range from clinical applications to fundamental research and is estimated at $17 B, and detecting these modifications is the fastest-growing application growing at an estimated 18% annually. The proposed protein-sequencing assay can characterize these protein modifications with 4-6 orders of magnitude greater sensitivity than current technologies. This sensitivity enables new classes of experiments in which only small samples are available (e.g. biopsies from living patients) or the target protein/modification is rare, and translates to substantial materials savings in all cases. The highly-sensitive characterization of proteins and their modifications will provide a new type of valuable quantitative data for scientists in industry and academic labs alike. This Small Business Technology Transfer (STTR) Phase I project will be to develop the single-molecule protein sequencing assay (fluorosequencing) for use by proteomics scientists to precisely quantify multiple phosphorylated sites on protein molecules. The best analytical technology today, mass-spectrometers, has an inherent limitation in identifying multiple (>2) closely spaced protein modifications and cannot produce accurate quantitative data if fewer than 10% of the proteins are modified at the particular amino acid. Better characterization and quantification of phosphorylation is recognized as a need by proteomics researchers. The project will explore the competitive ability of fluorosequencing to distinguish and quantify closely spaced modifications in multiple proteins. The project will also provide evidence for the capability of the technology to detect and quantify a single phosphorylated protein amongst 100 total proteins. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.