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

TOPIC 420 深度单细胞蛋白质组学综合平台 性能周期 2021年9月13日-2022年6月12日 FY21 第一期 SBIR

• 批准号: 10610297
• 负责人: RYAN KELLY
• 金额: $ 5.5万
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-13 至 2022-06-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10610297
• 关键词: Automation; Cancer Diagnostics; Cells; Computer software; Custom; Development; Hela Cells; Impairment; Malignant Neoplasms; Manuals; Measurement; Neoplasm Circulating Cells; Performance; Phase; Positioning Attribute; Preparation; Proteins; Proteome; Recovery; Reproducibility; Robotics; Sampling; Small Business Innovation Research Grant; Structure; System; Tissues; anticancer research; biological systems; cost; data quality; instrument; liquid chromatography mass spectrometry; nanolitre; nanolitre scale; open source; protein expression; single cell proteins; targeted treatment; tumor heterogeneity

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.

蛋白质是包括癌症在内的生物系统的关键功能和结构组分，并且是治疗剂的主要靶标。由于进行测量所需的大量组织，直接分析癌症研究和诊断的蛋白质表达的能力受到损害。这些批量分析掩盖了重要的肿瘤异质性，并且无法分析罕见但重要的细胞，如循环肿瘤细胞。我们最近开发了一种称为nanoPOTS的纳升级样品制备平台。当与超灵敏度分析相结合时，nanoPOTS能够从单细胞中分析数百至>1000种蛋白质，但目前涉及几个手动步骤和多个定制仪器，使其不适合广泛传播。我们将开发一个集成的机器人平台，完成纳升规模的样品制备和纳升规模的LC/MS分析接口。我们还将评估用于nanoPOTS处理的替代“单细胞”衬底材料和简化的一步处理工作流程。该系统的特点是可实现的蛋白质组覆盖率，再现性和吞吐量使用单一的HeLa细胞。数据质量将使用开源软件进行评估。我们希望在单个HeLa细胞中识别超过800种蛋白质，使系统在后续开发阶段实现全自动化并提高测量通量。