Targeted proteomics technology for accurate quantitative single-cell proteomics

摘要 肿瘤之间和肿瘤内广谱小细胞亚群的多组学表征 单个肿瘤的单细胞分辨率对于理解完整的疾病生物学至关重要。 此外，具有生物学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进行有效分析。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，低多重性），并且通常缺乏定量 精度基于质谱（MS）的靶向蛋白质组学已经成为广泛准确的蛋白质组学的替代方案。 量化然而，目前的单细胞MS只能相对定量约870种蛋白质， 单个哺乳动物细胞。单细胞MS在准确定量单细胞中存在三个主要挑战 蛋白质组学：1）单细胞的无效处理，2）MS灵敏度不足和样品通量低， 3）缺乏表征良好的通用内标物（UIS）。为了应对这些挑战，我们建议 建立单细胞质谱系统，用于单细胞蛋白质组的快速准确分析。可行性强 在我们最近在技术开发的许多方面的进展的支持下（例如，介绍“承运人” 有效处理少量细胞（包括单细胞）和发展破坏性MS的概念 技术，以提高MS检测灵敏度和特异性）以及我们在高， 分离液相色谱（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 1)建立super-SILAC（stable isotope labeling with amino acids in cell culture）作为蛋白质组载体 和UIS，2）将蛋白质组载体super-SILAC（cSILAC）并入样品制备工作流程， 单细胞的稳健处理，以及3）利用PNNL开发的尖端LC和MS技术 与超低流量LC分离集成，高效离子源（发射极阵列组合 技术和使用纳米电喷雾低于环境压力电离），以及超快高分辨率离子 迁移率分离显著提高MS灵敏度和样品通量。超级SILAC将 被表征为UIS，用于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方法有效准确地测定。96井 基于板的cSILAC制备和良好表征的UIS，新的单细胞MS系统预计将允许 用于快速准确定量单细胞中的大部分人类蛋白质组（约60%）， 每天约120个样本。我们预计，新的MS系统最终将成为一个方便的 它不仅是定量单细胞蛋白质组学的不可或缺的工具，也是非常小的常规分析的工具。 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重大贡献。

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.