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 多词的表征肿瘤之间的细胞和内部细胞的较小亚群 单细胞分辨率下的个别肿瘤对于了解完整疾病生物学至关重要。 此外，生物学上重要的临床标本以低量（例如，<10个肿瘤细胞）提供， 需要高级单细胞技术进行有效分析。但是，单细胞蛋白质组学技术 远远落后于其他OMICS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（例如低多重），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品 定量。但是，当前的单细胞MS只能允许对〜870蛋白的相对定量 单哺乳动物细胞。单细胞MS中有三个主要挑战，可准确定量单细胞 蛋白质组学：1）单细胞的无效处理，2）MS敏感性不足和样品吞吐量低， 3）缺乏特征良好的通用内标（UIS）。为了应对这些挑战，我们建议 开发一个单细胞MS系统，以快速准确地分析单细胞蛋白质组。可行性强烈 在我们最近在技术开发的许多方面取得进展的支持（例如，介绍“运营商” 有效处理包括单个细胞在内的少量细胞的概念，并开发破坏性的MS 提高MS检测敏感性和特异性的技术）以及我们在高级方面的丰富经验 分辨率液相色谱（LC）分离，用于敏感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 1）在细胞培养中建立超硅酸盐（稳定的同位素标记）作为蛋白质组载体 和UIS，2）将蛋白质组载体Super-SILAC（CSILAC）掺入样品制备工作流程中 单个细胞的鲁棒处理，以及3）利用pnnl开发的切割LC和MS技术 随着超流量LC分离的整合，高效离子源（发射极阵列的组合 纳米电喷雾的技术和次级压力电离）和超快高分辨率离子 迁移率分离，可显着提高MS灵敏度和样品吞吐量。 Super-Silac Will 用粗制肽标准标准为绝对定量的UI，其纯度将是成本的 有效地使用组合的灯笼标签和ICP-MS方法有效地确定。带96孔 基于板块的CSILAC制备和特征良好的UI，新的单细胞MS系统有望允许 为了快速准确地定量单个细胞中的大量人类蛋白质组（约60％） 〜每天120个样本。我们预计新的MS系统最终将成为方便 必不可少的工具不仅用于定量单细胞蛋白质组学，而且对于非常小的常规分析 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。