PFI-TT: A highly multiplexed readout system for single-molecule analysis

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The broader impact/commercial potential of this Partnerships for Innovation - Technology Translation (PFI-TT) project is to develop instrumentation that will enable deep profiling of cellular proteins. The methods will be made broadly applicable in fundamental biological research. The project’s central goal is to develop and validate instrumentation for studying proteomics – the collection of proteins constituting the molecular machinery underlying all life forms. Genes, the molecular precursors of cellular machinery, are templates encoding how proteins are constructed within cells. The genomics revolution has recently enabled decoding of the human genome. Similar advances in proteomics technology has not occurred since genomic studies rely upon gene amplification to making billions of identical copies to enable analysis en masse. No similar molecular amplification process exists for proteins, hence single-molecule analyses are key. The proposed research relies upon the collaborative efforts of researchers spanning physics, engineering, chemistry, biology, and mathematics – from both academia and industry. Component technologies for a feasible, commercializable instrument will be pursued. This project’s highly collaborative and rich research environment provides an opportunity for the graduate student who will be involved in this effort to gain important career skills. The proposed project will develop instrumentation to unravel the staggering complexity of protein biology. An individual mammalian cell comprises roughly 3B proteins, spanning about 7,000 unique types, with concentrations spanning 8 orders of magnitude – ranging from cellular proteins present with just a few copies, to those with expression levels of order 100M. In human blood serum this range is a 1000X greater, spanning 11 orders. Resolving the full spectrum of protein constituents is essential to fundamental questions in biology and medicine. Detailed analyses – at the population level, yet with single-molecule resolution – will stratify fine details lost in existing consensus-type approaches to proteomics that “average over” the protein population, thus providing information only about the most prevalent species. Critical processes in health and disease are often determined by only a few copies of a protein molecule within a cell. Accordingly, single-molecule resolution of proteins and protein complexes in the presence of an immense overabundance of the most prevalent species is essential.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.