Targeted proteomics technology for accurate quantitative single-cell proteomics

精准定量单细胞蛋白质组学的靶向蛋白质组学技术

• 批准号: 10096431
• 负责人: Tujin Shi
• 金额: $ 39.89万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-23 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10096431
• 关键词: Address; Adsorption; Amino Acids; Antibodies; Biological; Biology; Biomedical Research; Biopsy; Cancer Biology; Carrier Proteins; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Clinical; Collection; Complex; Coupled; Detection; Devices; Disease; Epidermal Growth Factor Receptor; Evolution; Genetic; Genomic Instability; Genomics; Genotype; Goals; Heterogeneity; Human; Immunoassay; Individual; Ions; Label; Lanthanoid Series Elements; Liquid Chromatography; MAP Kinase Gene; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Measurement; Messenger RNA; Methods; Movement; Pathway interactions; Peptides; Performance; Phenotype; Preparation; Proteins; Proteome; Proteomics; Recovery; Research; Resolution; Sampling; Sensitivity and Specificity; Signal Pathway; Source; Specificity; Specimen; Stable Isotope Labeling; Structure; Surface; System; Techniques; Technology; Variant; base; cancer heterogeneity; cost; detection sensitivity; experience; improved; insight; ion mobility; ion source; ionization; liquid chromatography mass spectrometry; minimally invasive; multiple omics; nano-electrospray; neoplastic cell; precision medicine; pressure; single cell analysis; single cell technology; surfactant; technology development; time of flight mass spectrometry; tool; transcriptome; transcriptomics; tumor; tumor heterogeneity; whole genome

ABSTRACT Multi-omics characterization of a broad spectrum of small subpopulations of cells between tumors and within individual tumors at the single-cell resolution is crucial to achieve understanding of a complete disease biology. Furthermore, biologically important clinical specimens are available in low quantity (e.g., <10 tumor cells), requiring advanced single-cell technologies for effective analysis. However, single-cell proteomics technologies are lagging far behind other omics technologies. Antibody-based immunoassays are used primarily for targeted single-cell proteomics, but they have inherent limitations (e.g., low multiplex), and generally lack quantitation accuracy. Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative for broad accurate quantification. However, current single-cell MS can only allow for relative quantification of ~870 proteins from single mammalian cells. There are three major challenges in single-cell MS for accurate quantitative single-cell proteomics: 1) ineffective processing of single cells, 2) insufficient MS sensitivity and low sample throughput, and 3) lacking well-characterized universal internal standard (UIS). To address these challenges, we propose to develop a single-cell MS system for rapid accurate analysis of single-cell proteome. The feasibility is strongly supported by our recent progress in many aspects of technology development (e.g., introducing the `carrier' concept for effective processing of small numbers of cells including single cells, and developing disruptive MS technologies to improve MS detection sensitivity and specificity) as well as our extensive experiences in high- resolution liquid chromatography (LC) separation for sensitive detection and targeted proteomics analysis for absolute quantification of signaling pathway proteins. The single-cell MS system will be developed through 1) establishing super-SILAC (stable isotope labeling with amino acids in cell culture) as both proteome carrier and UIS, 2) incorporation of proteome carrier super-SILAC (cSILAC) into the sample preparation workflow for robust processing of single cells, and 3) leveraging cutting-edging LC and MS technologies developed at PNNL with integration of ultralow-flow LC separation, high-efficiency ion source (the combination of an emitter array technology and sub-ambient pressure ionization with nanoelectrospray), and ultrafast high-resolution ion mobility separation for significantly improving both MS sensitivity and sample throughput. Super-SILAC will be characterized as UIS for absolute quantification with crude peptide standards, whose purity will be cost- effectively accurately determined using a combined lanthanide labeling and ICP-MS method. With 96-well plate-based cSILAC preparation and well-characterized UIS, the new single-cell MS system is expected to allow for rapid accurate quantification of a large fraction of human proteome (~60%) in single cells with ~120 samples per day. We anticipate that the new MS system will eventually become a convenient indispensable tool not only for quantitative single-cell proteomics but also for routine analysis of very small samples (e.g., rare cells). In turn, it will make substantial contributions to current biomedical research.

摘要 肿瘤之间和肿瘤内广泛分布的小亚群细胞的多组学特征 单个肿瘤的单细胞分辨率对于实现对完整疾病生物学的理解至关重要。 此外，具有生物重要性的临床标本数量较少(例如，10个肿瘤细胞)， 需要先进的单细胞技术来进行有效的分析。然而，单细胞蛋白质组学技术 远远落后于其他组学技术。以抗体为基础的免疫分析主要用于靶向。 单细胞蛋白质组学，但它们有固有的局限性(例如，低多样性)，并且通常缺乏定量 精确度。基于质谱学(MS)的靶向蛋白质组学已经成为广谱精确度的替代 量化。然而，目前的单细胞MS只能相对定量来自于 单个哺乳动物细胞。单细胞MS对准确定量单细胞有三个主要挑战 蛋白质组学：1)单细胞处理无效，2)MS灵敏度不足和样品吞吐量低， 3)缺乏有特色的通用内部标准(UIS)。为应付这些挑战，我们建议 建立单细胞蛋白质组快速准确分析的单细胞MS系统。可行性很强。 得到我们在技术发展的许多方面的最新进展的支持(例如，引入“航母” 有效处理少量细胞(包括单个细胞)和发展破坏性多发性硬化的概念 提高MS检测灵敏度和特异度的技术)，以及我们在高 用于灵敏检测和靶向蛋白质组学分析的拆分液相色谱(LC) 信号通路蛋白质的绝对定量。单细胞MS系统将通过以下方式开发 1)建立超稳定同位素标记的蛋白质组载体 和UIS，2)将蛋白质组载体Super-SILAC(CSILAC)纳入样品制备工作流程 稳健的单细胞处理，以及3)利用PNNL开发的尖端LC和MS技术 集成了超低流量LC分离、高效离子源(发射极阵列组合 技术和纳米电喷雾的亚常压电离)，以及超快高分辨率离子 流动分离可显著提高MS灵敏度和样品吞吐量。Super-SILAC将 被定性为UIS，用粗肽标准绝对定量，其纯度将是成本- 采用稀土标记和电感耦合等离子体质谱相结合的方法有效、准确地测定。有96口井 基于平板的cSILAC制备和良好的UIS特征，新的单细胞MS系统预计将允许 用于快速准确地定量单细胞中很大一部分人蛋白质组(~60%) 每天大约120个样品。我们预计，新的MS系统最终将成为一个方便的 不可缺少的工具，不仅是定量的单细胞蛋白质组学，也是非常小的 样本(例如，稀有细胞)。反过来，它将为当前的生物医学研究做出实质性贡献。