使用位置扫描肽微阵列分析蛋白质酪氨酸激酶特异性
Analysis of Protein Tyrosine Kinase Specificity Using Positional Scanning Peptide Microarrays.
作者信息
Deng Yang, Turk Benjamin E
机构信息
Department of Biomedical Engineering, Yale University, New Haven, CT, USA.
Department of Pharmacology, Yale University School of Medicine, SHM B395e, 333 Cedar Street, New Haven, CT, 06520, USA.
出版信息
Methods Mol Biol. 2016;1352:27-34. doi: 10.1007/978-1-4939-3037-1_3.
Protein tyrosine kinases phosphorylate their substrates within the context of specific consensus sequences surrounding the site of modification. We describe a peptide microarray approach to rapidly determine tyrosine kinase phosphorylation site motifs. This method uses a peptide library that systematically substitutes each of the amino acid residues at multiple positions surrounding a central tyrosine residue. Peptide substrates are synthesized as biotin conjugates for immobilization on avidin-coated slides. Following incubation of the slide with protein kinase and radiolabeled ATP, the relative extent of phosphorylation of each of the peptides is quantified by phosphor imaging. This method allows small quantities of kinase to be analyzed rapidly in parallel, facilitating analysis of large numbers of kinases.
蛋白质酪氨酸激酶在修饰位点周围的特定共有序列背景下使其底物磷酸化。我们描述了一种肽微阵列方法，用于快速确定酪氨酸激酶磷酸化位点基序。该方法使用一个肽库，该肽库系统地替换中心酪氨酸残基周围多个位置的每个氨基酸残基。肽底物被合成为生物素缀合物，用于固定在抗生物素蛋白包被的载玻片上。在载玻片与蛋白激酶和放射性标记的ATP孵育后，通过磷光成像对每个肽的磷酸化相对程度进行定量。该方法允许对少量激酶进行快速并行分析，便于对大量激酶进行分析。
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