Hilhorst Riet, Houkes Liesbeth, van den Berg Adriënne, Ruijtenbeek Rob
PamGene International BV, 5200 BJ 's-Hertogenbosch, The Netherlands.
Anal Biochem. 2009 Apr 15;387(2):150-61. doi: 10.1016/j.ab.2009.01.022. Epub 2009 Jan 21.
A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.
一种基于微阵列的混合测量、非放射性多重方法，结合实时检测，用于蛋白激酶A（PKA）的底物鉴定、检测方法开发、检测方法优化和动力学表征。肽阵列包含多达140种含丝氨酸/苏氨酸的肽或少量肽的浓度系列。与现有的单重检测相比，数据质量高，检测条件的变化和试剂消耗显著减少，并且由于可在4、12或96个阵列上同时监测磷酸化动力学，检测方法开发可以加速。结果表明，PKA可磷酸化许多含有已知PKA磷酸化位点的肽以及一些新底物。该酶在肽微阵列上的动力学行为以及AMP-PNP、星形孢菌素和PKA抑制肽的抑制机制与均相检测的数据相关性良好。使用这种多重设置，我们表明PKA的动力学参数和PKA抑制剂的效力会受到肽底物序列的影响。该技术能够在多重环境（如细胞或组织裂解物）中对激酶活性进行动力学监测。最后，这种高通量方法能够快速鉴定仍未表征的丝氨酸/苏氨酸激酶的肽底物。
