Espina Virginia, Mueller Claudius, Liotta Lance A
Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA.
Methods Mol Biol. 2011;785:23-43. doi: 10.1007/978-1-61779-286-1_3.
Phosphorylated proteins reflect the activity of specific cell signaling nodes in biological kinase protein networks. Cell signaling pathways can be either activated or deactivated depending on the phosphorylation state of the constituent proteins. The state of these kinase pathways reflects the in vivo activity of the cells and tissue at any given point in time. As such, cell signaling pathway information can be extrapolated to infer which phosphorylated proteins/pathways are driving an individual tumor's growth. Reverse phase protein microarrays (RPMAs) are a sensitive and precise platform that can be applied to the quantitative measurement of hundreds of phosphorylated signal proteins from a small sample of tissue. Pre-analytical variability originating from tissue procurement and preservation may cause significant variability and bias in downstream molecular analysis. Depending on the ex vivo delay time in tissue processing, and the manner of tissue handling, protein biomarkers such as signal pathway phosphoproteins will be elevated or suppressed in a manner that does not represent the biomarker levels at the time of excision. Consequently, assessment of the state of these kinase networks requires stabilization, or preservation, of the phosphoproteins immediately post-tissue procurement. We have employed RPMA analysis of phosphoproteins to study the factors influencing stability of phosphoproteins in tissue following procurement. Based on this analysis we have established tissue procurement guidelines for clinical research with an emphasis on quantifying phosphoproteins by RPMA.
磷酸化蛋白反映了生物激酶蛋白网络中特定细胞信号节点的活性。细胞信号通路可根据组成蛋白的磷酸化状态被激活或失活。这些激酶通路的状态反映了细胞和组织在任何给定时间点的体内活性。因此，可以推断细胞信号通路信息，以确定哪些磷酸化蛋白/通路驱动个体肿瘤的生长。反相蛋白质微阵列（RPMA）是一个灵敏且精确的平台，可用于对少量组织样本中的数百种磷酸化信号蛋白进行定量测量。源自组织采集和保存的分析前变异性可能会在下游分子分析中导致显著的变异性和偏差。根据组织处理中的体外延迟时间以及组织处理方式，诸如信号通路磷酸化蛋白等蛋白质生物标志物会以不代表切除时生物标志物水平的方式升高或降低。因此，评估这些激酶网络的状态需要在组织采集后立即稳定或保存磷酸化蛋白。我们采用了对磷酸化蛋白的RPMA分析来研究影响采集后组织中磷酸化蛋白稳定性的因素。基于此分析，我们制定了临床研究的组织采集指南，重点是通过RPMA对磷酸化蛋白进行定量。
