Robust ultra-high sensitivity proteomic technologies for limited samples

适用于有限样品的稳健超高灵敏度蛋白质组技术

• 批准号: 10202666
• 负责人: Alexander R. Ivanov
• 金额: $ 41.18万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10202666
• 关键词: Biological; Biological Models; Biological Process; Biomedical Research; Cancer cell line; Cell Differentiation process; Cells; Characteristics; Clinical; Complex; Data Analyses; Development; Diagnostic; Diagnostics Research; Disease; Hematopoietic stem cells; Immune; Individual; Knowledge; Lead; Ligands; Liquid substance; Malignant Neoplasms; Malignant neoplasm of ovary; Mass Spectrum Analysis; Methods; Microscopic; Molecular; Neoplasm Circulating Cells; Peptides; Pharmaceutical Preparations; Phase; Play; Population; Post-Translational Protein Processing; Preparation; Process; Prognostic Marker; Proteins; Proteome; Proteomics; Research; Role; STAT3 gene; Sampling; Specimen; Techniques; Technology; X-Ray Crystallography; Yeasts; base; clinically relevant; data acquisition; diagnostic biomarker; innovation; insight; liquid biopsy; new therapeutic target; non-Native; novel; novel strategies; personalized medicine; prognostic; protein complex; public health relevance; research and development; response biomarker; screening; targeted biomarker; technology development; therapeutic target; therapy development; transcription factor; yeast two hybrid system

PROJECT SUMMARY The majority of highly diverse biological processes are enabled through proteins, protein post-translational modifications/proteoforms, protein interactions, PIs (e.g., protein-protein, PPIs, protein- ligand, PLIs), and aberrations of abundances, activities, functions, and integrity of such interactions can lead to severe diseases, including cancer. Furthermore, disruption of these protein-based characteristics by novel targeted therapies can be an important biomarker for the response to these drugs in personalized medicine approaches. Clinical and biological specimens are often available in limited amounts, which greatly hampers the progress in diagnostics, therapy development, and biomedical research. Microbiopsy and liquid biopsies containing rare cell populations such as circulating tumor cells, hematopoietic stem cells (HSCs) and immune cells may contain only low thousands or hundreds of cells and be heterogeneous. Traditional techniques to study proteomic profiles, proteoforms, protein complexes, and PPIs (e.g., conventional proteomics, NMR, X-ray crystallography, yeast two-hybrid screening and immunoaffinity purification (IP) followed by mass spectrometry (MS)) cannot be readily used for the analysis of small cell populations, microscopic clinical samples and individual cells mainly due to limitations in sensitivity. Therefore, many biological and clinically relevant studies are not undertaken because of the lack of technology for such low level samples. Here, we propose to develop analytical platforms that will enable high sensitivity analysis of scarce samples at the level of digests, intact proteoforms, and native complexes. This task will demand the development of novel approaches in sample preparation, ulra-low flow liquid phase separations interfaced with MS, MS data acquisition, and data analysis. Developing such novel methods for thorough profiling of microscale samples and integrating them in innovative “plug-and-play” automated platforms capable of efficient and high sensitivity characterization of intact proteoforms, protein complexes and PTMs by MS will be highly desirable for gaining biological insights into molecular mechanisms of the disease and discovery of therapeutic targets and biomarkers for diagnostic and prognostic purposes. The developed platforms will be evaluated using well- controlled model systems and applied in the most clinically relevant settings to examine (1) model systems for cell differentiation and activation; (2) the interactome and biological role of STAT3, the transcription factor which is aberrantly activated in the vast majority of ovarian cancer cell lines and primary samples; and (2) MHC-associated neontigenic peptides.

项目概要 大多数高度多样化的生物过程是通过蛋白质、蛋白质翻译后实现的 修饰/蛋白质形式、蛋白质相互作用、PI（例如蛋白质-蛋白质、PPI、蛋白质-配体、PLI）以及 这种相互作用的丰度、活性、功能和完整性的异常可能导致严重的疾病， 此外，新的靶向疗法会破坏这些基于蛋白质的特征。 可能是个性化临床方法中对这些药物反应的重要生物标志物。 而且生物标本的数量往往有限，这极大地阻碍了研究的进展 诊断、治疗开发和生物医学研究，包含稀有的活检和液体活检。 循环肿瘤细胞、造血干细胞 (HSC) 和免疫细胞等细胞群可能 仅包含数千或数百个细胞，并且是异质的传统研究技术。 蛋白质组图谱、蛋白质形式、蛋白质复合物和 PPI（例如，传统蛋白质组学、NMR、X 射线 晶体学、酵母双杂交筛选和免疫亲和纯化 (IP)，然后进行质谱分析 (MS)) 不能轻易用于分析小细胞群、显微临床样本和 主要由于个体细胞敏感性的限制，因此有许多生物学和临床相关的研究。 由于缺乏处理如此低水平样品的技术，因此没有进行。在这里，我们建议 开发分析平台，能够对稀缺样品进行高灵敏度分析 这项任务需要开发新的消化物、完整的蛋白质形式和天然复合物。 样品制备、超低流量液相分离与 MS、MS 数据接口的方法 开发这种新颖的方法来彻底分析微型样品。 并将它们集成到具有高效和高灵敏度的创新“即插即用”自动化平台中 通过 MS 对完整的蛋白质形式、蛋白质复合物和 PTM 进行表征对于获得 对疾病分子机制的生物学见解以及治疗靶点的发现和 用于诊断和预后目的的生物标志物将使用良好的评估。 受控模型系统并应用于最临床相关的环境中以检查（1）模型系统 细胞分化和激活；（2）转录因子STAT3的相互作用和生物学作用 它在绝大多数卵巢癌细胞系和原代样本中被异常激活；以及 (2) MHC 相关新生肽。