Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach.
Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach.
复制标题
使用蛋白质组肽库 (ProPeL) 方法表征蛋白激酶底物特异性。
DOI：
10.1002/cpch.38
复制
发表时间：
2018
影响因子：
--
通讯作者：
Schwartz,Daniel
中科院分区：
文献类型：
--
作者：
Lubner,JoshuaM;Balsbaugh,JeremyL;Church,GeorgeM;Chou,MichaelF;Schwartz,Daniel
鉴定蛋白激酶底物特异性基序是阐明蛋白激酶信号级联的重要一步。这里描述的方案使用细菌系统来评估活体内的激酶特异性基序，而不需要放射性的ATP。将感兴趣的人的激酶克隆到异源细菌表达载体中，并允许其磷酸化E。在体内，与其内源底物偏好一致。细胞被裂解，细菌蛋白质被消化成多肽，并使用块状二氧化钛进行磷浓缩。用串联质谱仪鉴定聚集的磷酸肽，并使用pLogo可视化工具进行生物信息学分析。PROPEL方法可以对野生型激酶特异性基序进行详细的表征，鉴定由于激酶突变引起的特异性漂移，并评估激酶残基的结构-功能关系。2018年John Wiley&Sons，Inc.
Characterizing protein kinase substrate specificity motifs represents a powerful step in elucidating kinase‐signaling cascades. The protocol described here uses a bacterial system to evaluate kinase specificity motifsin vivo, without the need for radioactive ATP. The human kinase of interest is cloned into a heterologous bacterial expression vector and allowed to phosphorylateE. coliproteinsin vivo, consistent with its endogenous substrate preferences. The cells are lysed, and the bacterial proteins are digested into peptides and phosphoenriched using bulk TiO2. The pooled phosphopeptides are identified by tandem mass spectrometry, and bioinformatically analyzed using the pLogo visualization tool. The ProPeL approach allows for detailed characterization of wildtype kinase specificity motifs, identification of specificity drift due to kinase mutations, and evaluation of kinase residue structure‐function relationships. © 2018 by John Wiley & Sons, Inc.