Phosphospecific proteolysis for mapping sites of protein phosphorylation
Phosphospecific proteolysis for mapping sites of protein phosphorylation
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DOI：
10.1038/nbt863
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发表时间：
2003-09-01
影响因子：
46.9
通讯作者：
Shokat, KM
中科院分区：
文献类型：
--
作者：
Knight, ZA;Schilling, B;Shokat, KM
蛋白质磷酸化是细胞内信息传递的主要机制，当前蛋白质组学研究的一个主要目标是绘制一幅描述所有蛋白质磷酸化位点的系统层面图谱。近期的研究工作集中在开发用于富集和定量磷酸肽的技术上。磷酸化位点的鉴定通常依赖串联质谱对单个肽段进行测序。在此我们描述一种磷酸肽图谱绘制方法，该方法能够利用一种识别磷酸化位点的蛋白酶直接对蛋白质序列进行检测。这种方法的关键在于将磷酸丝氨酸和磷酸苏氨酸残基选择性地化学转化为赖氨酸类似物（分别为氨乙基半胱氨酸和β - 甲基氨乙基半胱氨酸）。然后用一种赖氨酸特异性蛋白酶切割氨乙基半胱氨酸修饰的肽段，以绘制磷酸化位点图谱。一个封闭步骤可实现单位点切割，并且将该反应应用于固相有助于在一个步骤中实现磷酸肽的富集和修饰。
Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a system-level map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and beta-methylaminoethylcysteine, respectively). Aminoethylcysteine-modified peptides are then cleaved with a lysine-specific protease to map sites of phosphorylation. A blocking step enables single-site cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step.