EO14042-TOPIC 420 INTEGRATED PLATFORM FOR IN-DEPTH SINGLE-CELL PROTEOMICSPERIOD OF PERFORMANCE SEPTEMBER 13, 2021-AUGUST 21, 2022FY21 PHASE I SBIR

Proteins are key functional and structure components of biological systems including cancers, and are the primary targets of therapeutics. The ability to directly profile protein expression for cancer research and diagnostics has been impaired by the large amounts of tissue required to perform a measurement. These bulk analyses obscure important tumor heterogeneity and are unable to profile rare but important cells such as circulating tumor cells. We have recently developed a nanoliter-scale sample preparation platform termed nanoPOTS. When combined with ultrasensitive analysis, nanoPOTS enables profiling of hundreds to rt1000 proteins from single cells but currently involves several manual steps and multiple custom instruments that make it unsuitable for broad dissemination. We will develop an integrated robotic platform that accomplishes both nanoliter-scale sample preparation and nanoliter-scale interfacing with LC/MS analysis. We will also evaluate alternative ‘nanowell’ substrate materials for nanoPOTS processing and a simplified one-step processing workflow. The system will be characterized in terms of achievable proteome coverage, reproducibility and throughput using single HeLa cells. Data quality will be assessed using open-source software. We expect to identify rt800 proteins in single HeLa cells, positioning the system for full automation and increased measurement throughput during the subsequent phase of development.