TOPIC 420 INTEGRATED PLATFORM FOR IN-DEPTH SINGLE-CELL PROTEOMICSPERIOD OF PERFORMANCE SEPTEMBER 13, 2021-JUNE 12, 2022FY21 PHASE I SBIR

蛋白质是包括癌症在内的生物系统的关键功能和结构成分，也是治疗学的主要靶点。直接分析用于癌症研究和诊断的蛋白质表达的能力因进行测量所需的大量组织而受到损害。这些整体分析掩盖了重要的肿瘤异质性，也无法描绘罕见但重要的细胞，如循环中的肿瘤细胞。我们最近开发了一个纳升尺度的样品制备平台，称为NanPOTS。当与超灵敏的分析相结合时，NanPOTS能够从单个细胞中分析数百到&gt；1000个蛋白质，但目前需要几个手动步骤和多个定制仪器，这使得它不适合广泛传播。我们将开发一个集成的机器人平台，完成纳升尺度的样品制备和纳升尺度的接口与LC/MS分析。我们还将评估用于纳米POTS处理和简化的一步处理工作流程的可替代的“Nanowell”衬底材料。该系统将在可实现的蛋白质组覆盖率、重复性和使用单个HeLa细胞的吞吐量方面进行表征。将使用开源软件对数据质量进行评估。我们希望在单个HeLa细胞中识别&GT；800蛋白质，将系统定位为全自动化，并在随后的开发阶段增加测量吞吐量。

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 >1000 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 >800 proteins in single HeLa cells, positioning the system for full automation and increased measurement throughput during the subsequent phase of development.