Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital , Central South University , Changsha , Hunan 410008 , People's Republic of China.
Cancer Biomarkers Research Group, Division of Cancer Prevention , National Cancer Institute , Bethesda , Maryland 20892 , United States.
Anal Chem. 2019 Jan 15;91(2):1441-1451. doi: 10.1021/acs.analchem.8b04258. Epub 2018 Dec 28.
Heterogeneity in composition is inherent in all cell populations, even those containing a single cell type. Single-cell proteomics characterization of cell heterogeneity is currently achieved by antibody-based technologies, which are limited by the availability of high-quality antibodies. Herein we report a simple, easily implemented, mass spectrometry (MS)-based targeted proteomics approach, termed cLC-SRM (carrier-assisted liquid chromatography coupled to selected reaction monitoring), for reliable multiplexed quantification of proteins in low numbers of mammalian cells. We combine a new single-tube digestion protocol to process low numbers of cells with minimal loss together with sensitive LC-SRM for protein quantification. This single-tube protocol builds upon trifluoroethanol digestion and further minimizes sample losses by tube pretreatment and the addition of carrier proteins. We also optimized the denaturing temperature and trypsin concentration to significantly improve digestion efficiency. cLC-SRM was demonstrated to have sufficient sensitivity for reproducible detection of most epidermal growth factor receptor (EGFR) pathway proteins expressed at levels ≥30 000 and ≥3000 copies per cell for 10 and 100 mammalian cells, respectively. Thus, cLC-SRM enables reliable quantification of low to moderately abundant proteins in less than 100 cells and could be broadly useful for multiplexed quantification of important proteins in small subpopulations of cells or in size-limited clinical samples. Further improvements of this method could eventually enable targeted single-cell proteomics when combined with either SRM or other emerging ultrasensitive MS detection.
细胞群体的组成存在异质性，即使是包含单一细胞类型的群体也是如此。单细胞蛋白质组学对细胞异质性的特征描述目前是通过抗体技术实现的，但这种技术受到高质量抗体可用性的限制。在此，我们报告了一种简单、易于实施的基于质谱（MS）的靶向蛋白质组学方法，称为 cLC-SRM（载体辅助液相色谱与选择反应监测相结合），可用于可靠地对低数量哺乳动物细胞中的蛋白质进行多重定量。我们将一种新的单管消化方案与敏感的 LC-SRM 结合起来，用于对少量细胞进行处理，以实现最小化的损失，从而进行蛋白质定量。这种单管方案建立在三氟乙醇消化的基础上，并通过管预处理和添加载体蛋白进一步最小化样品损失。我们还优化了变性温度和胰蛋白酶浓度，以显著提高消化效率。cLC-SRM 的灵敏度足以重复检测到大多数表皮生长因子受体（EGFR）通路蛋白，这些蛋白的表达水平≥30000 时，10 个哺乳动物细胞中每个细胞的拷贝数≥3000；表达水平≥30000 时，100 个哺乳动物细胞中每个细胞的拷贝数≥3000。因此，cLC-SRM 能够可靠地对低丰度到中丰度的蛋白质进行定量，其数量少于 100 个细胞，并且对于在细胞的小亚群或大小有限的临床样本中对重要蛋白质进行多重定量可能具有广泛的用途。当与选择反应监测（SRM）或其他新兴的超灵敏 MS 检测方法结合使用时，该方法的进一步改进最终可能实现靶向单细胞蛋白质组学。
