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.

抽象的 肿瘤之间和内部的广泛小细胞亚群的多组学表征 单细胞分辨率的单个肿瘤对于了解完整的疾病生物学至关重要。 此外，生物学上重要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。基于抗体的免疫测定主要用于靶向 单细胞蛋白质组学，但它们具有固有的局限性（例如，多重性低），并且通常缺乏定量 准确性。基于质谱 (MS) 的靶向蛋白质组学已成为广泛准确的替代方法 量化。然而，目前的单细胞 MS 只能对约 870 种蛋白质进行相对定量 单个哺乳动物细胞。单细胞质谱准确定量单细胞面临三大挑战 蛋白质组学：1) 单细胞处理无效，2) MS 灵敏度不足且样品通量低， 3) 缺乏特征明确的通用内标 (UIS)。为了应对这些挑战，我们建议 开发单细胞 MS 系统，用于快速准确地分析单细胞蛋白质组。可行性很强 我们最近在技术开发的许多方面取得的进展（例如，引入“载体” 有效处理少量细胞（包括单细胞）和开发破坏性 MS 的概念 提高 MS 检测灵敏度和特异性的技术）以及我们在高 分辨率液相色谱 (LC) 分离，用于灵敏检测和靶向蛋白质组学分析 信号通路蛋白的绝对定量。单细胞 MS 系统将通过以下方式开发： 1）建立super-SILAC（细胞培养物中氨基酸的稳定同位素标记）作为蛋白质组载体 和 UIS，2) 将蛋白质组载体 super-SILAC (cSILAC) 纳入样品制备工作流程中 单细胞的稳健处理，以及 3) 利用 PNNL 开发的尖端 LC 和 MS 技术 集成超低流量 LC 分离、高效离子源（发射器阵列的组合） 技术和亚环境压力电离（纳电喷雾），以及超快高分辨率离子 迁移率分离可显着提高 MS 灵敏度和样品通量。超级SILAC将 被表征为UIS，用于使用粗肽标准品进行绝对定量，其纯度将是成本- 使用组合的镧系元素标记和 ICP-MS 方法可以有效准确地测定。带96孔 基于板的 cSILAC 制备和充分表征的 UIS，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大部分人类蛋白质组 (~60%) 每天约 120 个样本。我们预计新的 MS 系统最终将成为一种方便的 不仅是定量单细胞蛋白质组学不可或缺的工具，也是非常小的常规分析的必备工具 样品（例如稀有细胞）。反过来，它将为当前的生物医学研究做出重大贡献。