Knight Zachary A, Schilling Birgit, Row Richard H, Kenski Denise M, Gibson Bradford W, Shokat Kevan M
Program in Chemistry and Chemical Biology, University of California, San Francisco, California 94143-0446, USA.
Nat Biotechnol. 2003 Sep;21(9):1047-54. doi: 10.1038/nbt863. Epub 2003 Aug 17.
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.
蛋白质磷酸化是细胞中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生成一个系统水平的图谱，描述蛋白质磷酸化的所有位点。最近的研究工作集中在开发富集和定量磷酸肽的技术。磷酸化位点的鉴定通常依赖于串联质谱对单个肽段进行测序。在此，我们描述了一种磷酸肽图谱绘制方法，该方法使得用识别磷酸化位点的蛋白酶直接询问蛋白质序列成为可能。这种方法的关键是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（分别为氨基乙基半胱氨酸和β-甲基氨基乙基半胱氨酸）。然后用赖氨酸特异性蛋白酶切割氨基乙基半胱氨酸修饰的肽段，以绘制磷酸化位点。一个封闭步骤能够实现单一位点切割，并且将该反应应用于固相有助于一步实现磷酸肽的富集和修饰。
