Protein kinase substrate identification on functional protein arrays.
Protein kinase substrate identification on functional protein arrays.
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DOI：
10.1186/1472-6750-8-22
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发表时间：
2008-02-28
影响因子：
3.5
通讯作者：
Schweitzer B
中科院分区：
文献类型：
--
作者：
Meng L;Michaud GA;Merkel JS;Zhou F;Huang J;Mattoon DR;Schweitzer B
在过去的十年中，激酶已经成为许多不同疾病的有吸引力的治疗靶点，并且制药界的许多高通量筛选努力都是针对发现调节激酶功能的化合物。系统生物学方法的新兴实用性要求开发适合蛋白质组规模实验的多重工具，以取代低通量技术，例如用于蛋白质磷酸化研究的质谱法。最近，一种新的方法，用于确定底物的蛋白激酶已应用功能蛋白质阵列的小型化格式来表征磷酸化的候选蛋白质底物在一个单一的实验中的数千个。该方法包括将溶液中的蛋白激酶添加到固定化蛋白阵列中，以使用高灵敏度放射性检测和命中识别算法来识别底物。迄今为止，基于蛋白质阵列的激酶底物鉴定的最佳性能所需的因素尚未描述。在目前的研究中，我们已经进行了详细的表征的蛋白质阵列为基础的激酶底物鉴定方法，包括时间，缓冲液成分，和蛋白质浓度的影响的结果检查。将蛋白质阵列方法与用于评估底物磷酸化的基于标准溶液的测定进行比较，并且观察到大于80%的相关性。这里展示的结果表明，蛋白激酶的新底物可以从包含数千种人类蛋白质的阵列中快速识别，为蛋白激酶功能提供新的线索。此外，开发并应用了合并-去卷积策略，该策略增强了特定激酶-底物关系的表征并降低了试剂消耗。功能性蛋白质微阵列是一种重要的新工具，它能够对蛋白质磷酸化进行多重分析，因此可以用于鉴定新的激酶底物。将这项技术与细胞信号传导的系统生物学方法相结合，将有助于揭示我们对这类重要酶的理解的新层面。
Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Functional protein microarrays are an important new tool that enables multiplex analysis of protein phosphorylation, and thus can be utilized to identify novel kinase substrates. Integrating this technology with a systems biology approach to cell signalling will help uncover new layers in our understanding of this essential class of enzymes.
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