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Quantification of ERK Kinase Activity in Biological Samples Using Differential Sensing.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2019-12-16 , DOI: 10.1021/acschembio.9b00580 Diana Zamora-Olivares 1, 2 , Tamer S Kaoud 3, 4 , Lingyu Zeng 5 , Jacey R Pridgen 3 , Deborah L Zhuang 2 , Yakndara E Ekpo 2 , Jessica R Nye 2 , Mitchell Telles 2 , Eric V Anslyn 1 , Kevin N Dalby 3, 6
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2019-12-16 , DOI: 10.1021/acschembio.9b00580 Diana Zamora-Olivares 1, 2 , Tamer S Kaoud 3, 4 , Lingyu Zeng 5 , Jacey R Pridgen 3 , Deborah L Zhuang 2 , Yakndara E Ekpo 2 , Jessica R Nye 2 , Mitchell Telles 2 , Eric V Anslyn 1 , Kevin N Dalby 3, 6
Affiliation
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The understanding of complex biological systems requires an ability to evaluate interacting networks of genes, proteins, and cellular reactions. Enabling technologies that support the rapid quantification of these networks will facilitate the development of biological models and help to identify treatment targets and to assess treatment plans. The biochemical process of protein phosphorylation, which underlies almost all aspects of cell signaling, is typically evaluated by immunoblotting procedures (Western blot) or more recently proteomics procedures, which provide qualitative estimates of the concentration of proteins and their modifications in cells. However, protein modifications are difficult to correlate with activity, and while immunoblotting and proteomics approaches have the potential to be quantitative, they require a complex series of steps that diminish reproducibility. Here, a complementary approach is presented that allows for the rapid quantification of a protein kinase activity in cell lysates and tumor samples. Using the activity of cellular ERK (extracellular signal-regulated kinase) as a test case, an array sensing approach that utilizes a library of differential peptide-based biosensors and chemometric tools was used to rapidly quantify nanograms of active ERK in micrograms of unfractionated cell lysates and tumor extracts. This approach has the potential both for high-throughput and for quantifying the activities of multiple protein kinases in a single biological sample. The critical advantages of this differential sensing approach over others are that it removes the need for the addition of exogenous inhibitors to suppress the activities of major off-target kinases and allows us to quantitate the amount of active kinase in tested samples rather than measuring the changes in its activity upon induction or inhibition.
中文翻译：
使用差分传感定量生物样品中的ERK激酶活性。
对复杂生物系统的理解要求具有评估基因，蛋白质和细胞反应相互作用网络的能力。支持对这些网络进行快速量化的支持技术将促进生物学模型的开发，并有助于确定治疗目标和评估治疗计划。蛋白质磷酸化的生化过程几乎是细胞信号传导所有方面的基础，通常通过免疫印迹法（蛋白质印迹法）或更近期的蛋白质组学方法进行评估，这些方法可对蛋白质浓度及其在细胞中的修饰进行定性评估。但是，蛋白质修饰很难与活性相关联，尽管免疫印迹和蛋白质组学方法具有定量的潜力，他们需要一系列复杂的步骤，从而降低了可重复性。在这里，提出了一种补充方法，可以快速定量细胞裂解液和肿瘤样品中的蛋白激酶活性。使用细胞ERK（细胞外信号调节激酶）的活性作为测试案例，使用了基于差分肽基生物传感器和化学计量工具的阵列传感方法，以微克级分的细胞裂解液快速定量分析了活性ERK的纳克数。和肿瘤提取物。这种方法具有高通量和定量单个生物样品中多种蛋白激酶活性的潜力。
更新日期：2019-12-17
中文翻译：
使用差分传感定量生物样品中的ERK激酶活性。
对复杂生物系统的理解要求具有评估基因，蛋白质和细胞反应相互作用网络的能力。支持对这些网络进行快速量化的支持技术将促进生物学模型的开发，并有助于确定治疗目标和评估治疗计划。蛋白质磷酸化的生化过程几乎是细胞信号传导所有方面的基础，通常通过免疫印迹法（蛋白质印迹法）或更近期的蛋白质组学方法进行评估，这些方法可对蛋白质浓度及其在细胞中的修饰进行定性评估。但是，蛋白质修饰很难与活性相关联，尽管免疫印迹和蛋白质组学方法具有定量的潜力，他们需要一系列复杂的步骤，从而降低了可重复性。在这里，提出了一种补充方法，可以快速定量细胞裂解液和肿瘤样品中的蛋白激酶活性。使用细胞ERK（细胞外信号调节激酶）的活性作为测试案例，使用了基于差分肽基生物传感器和化学计量工具的阵列传感方法，以微克级分的细胞裂解液快速定量分析了活性ERK的纳克数。和肿瘤提取物。这种方法具有高通量和定量单个生物样品中多种蛋白激酶活性的潜力。
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