Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells
Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells
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DOI：
10.1158/1535-7163.mct-06-0334
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发表时间：
2006-10-01
影响因子：
5.7
通讯作者：
Kornblau, Steven M.
中科院分区：
文献类型：
--
作者：
Tibes, Raoul;Qiu, YiHua;Kornblau, Steven M.
蛋白质组学有可能通过对蛋白质表达水平和激活状态的全面分析来提供癌症发病机制的答案并指导靶向治疗。实现这一潜力需要开发新的、快速的、高通量的技术来对患者样本进行蛋白质阵列分析，以及开发新的分析技术来解释它们。在此，我们描述了使用反相蛋白阵列（RPPA）分析原发性急性髓细胞白血病样本以及白血病和正常干细胞的验证和稳健性。在这份报告中，我们表明，阵列打印，检测，扩增和染色精度非常高，可重复，他们与传统的蛋白质印迹。使用相同样品在相同和/或单独阵列上的重复，或使用从相同起始样品制备的单独蛋白质样品，阵列内和阵列间再现性极高。在阵列设置内，未检测到蛋白质信号强度的统计学显著差异。在检测延迟处理和制备多个冻融样品的实验中，维持了活化状态（磷酸化）。在蛋白质表达的差异可以可靠地检测到少至三个细胞蛋白质当量。从罕见的正常和白血病干细胞群体中制备的RPPA成功完成，并显示出与大量细胞群体的差异。示例显示了RPPA是如何理想地适用于目标识别，验证和药物发现的大规模分析。总之，RPPA是一种高度可靠、可重复、高通量的系统，可以对原代急性髓性白血病细胞、细胞系和人类干细胞中的蛋白表达和磷酸化状态进行快速、大规模的蛋白质组学分析。
Proteomics has the potential to provide answers in cancer pathogenesis and to direct targeted therapy through the comprehensive analysis of protein expression levels and activation status. The realization of this potential requires the development of new, rapid, high-throughput technologies for performing protein arrays on patient samples, as well as novel analytic techniques to interpret them. Herein, we describe the validation and robustness of using reverse phase protein arrays (RPPA) for the analysis of primary acute myelogenous leukemia samples as well as leukemic and normal stem cells. In this report, we show that array printing, detection, amplification, and staining precision are very high, reproducible, and that they correlate with traditional Western blotting. Using replicates of the same sample on the same and/or separate arrays, or using separate protein samples prepared from the same starting sample, the intra- and interarray reproducibility was extremely high. No statistically significant difference in protein signal intensities could be detected within the array setups. The activation status (phosphorylation) was maintained in experiments testing delayed processing and preparation from multiple freeze-thawed samples. Differences in protein expression could reliably be detected in as few as three cell protein equivalents. RPPA prepared from rare populations of normal and leukemic stem cells were successfully done and showed differences from bulk populations of cells. Examples show how RPPAs are ideally suited for the large-scale analysis of target identification, validation, and drug discovery. In summary, RPPA is a highly reliable, reproducible, high-throughput system that allows for the rapid large-scale proteomic analysis of protein expression and phosphorylation state in primary acute myelogenous leukemia cells, cell lines, and in human stem cells.