Department of Biomedical Engineering, Oregon Health &Science University, Portland OR 97201, USA.
OHSU Center for Spatial Systems Biomedicine, Oregon Health &Science University, Portland OR 97201, USA.
Sci Rep. 2016 Jun 20;6:28163. doi: 10.1038/srep28163.
Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation. We implement this digitized proteomic approach in an integrated imaging platform, the single cell-quantum dot platform (SC-QDP), to execute sensitive single cell phosphoquantitation in response to multiple drug treatment conditions and using limited primary patient material. The SC-QDP: 1) identified pAKT and pERK phospho-heterogeneity and insensitivity in individual leukemia cells treated with a multi-drug panel of FDA-approved kinase inhibitors, and 2) revealed subpopulations of drug-insensitive CD34+ stem cells with high pCRKL and pSTAT5 signaling in chronic myeloid leukemia patient blood samples. This ultrasensitive digitized protein detection approach is valuable for uncovering subtle but important differences in signaling, drug insensitivity, and other key cellular processes amongst single cells.
许多重要的信号和调节蛋白的表达丰度低，难以在单细胞中进行测量。我们报告了一种分子成像方法，通过数字化、离散计数纳米颗粒标记的蛋白质来定量蛋白质水平。数字化蛋白质计数可对单细胞中的蛋白质进行超灵敏的分子检测，优于定量总弥散荧光的传统方法，并可大大提高蛋白质定量的准确性。我们在单细胞量子点平台（SC-QDP）这一集成成像平台中实施了这种数字化蛋白质组学方法，以在响应多种药物处理条件和使用有限的原始患者材料的情况下执行敏感的单细胞磷酸化定量。SC-QDP：1）鉴定了经 FDA 批准的激酶抑制剂多药物组处理的单个白血病细胞中 pAKT 和 pERK 磷酸化异质性和不敏感性，2）揭示了慢性髓系白血病患者血液样本中 CD34+ 干细胞中具有高 pCRKL 和 pSTAT5 信号的药物不敏感亚群。这种超灵敏的数字化蛋白质检测方法对于揭示单细胞中信号、药物不敏感性和其他关键细胞过程中的细微但重要的差异非常有价值。
