Selective extraction and characterization of a histidine-phosphorylated peptide using immobilized copper(II) ion affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Selective extraction and characterization of a histidine-phosphorylated peptide using immobilized copper(II) ion affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
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DOI：
10.1021/ac026340f
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发表时间：
2003-04-01
影响因子：
7.4
通讯作者：
Ross, ARS
中科院分区：
文献类型：
--
作者：
Napper, S;Kindrachuk, J;Ross, ARS
磷酸化是原核和真核细胞中参与调节酶活性和介导信号转导的主要翻译后修饰。在质谱分析之前选择性富集磷酸化肽有助于鉴定磷酸化蛋白质，确定特定的磷酸化残基，并表征磷酸化发生的条件。已经使用固定化金属离子亲和色谱法为含有形成磷酸酯的残基（例如丝氨酸和苏氨酸）的肽建立了这样的方案。尽管组氨酸磷酸化在双组分信号转导途径中的重要性，但由于这些修饰的不稳定性和未磷酸化组氨酸与固定化金属离子相互作用的倾向，含有磷酸化组氨酸（P-His）残基的肽的类似方案已被证明是难以捉摸的。我们描述了一种使用固定化铜（II）离子和一次性金属螯合吸头（ZipTip（MC），Millipore）选择性提取含P-His肽的方法。该方法取决于磷酸基团和固定化铜（II）离子之间的pH依赖性相互作用。基质辅助激光解吸/电离飞行时间质谱与postsource衰减确认的身份和磷酸化状态的提取的肽。不保留含有非磷酸化组氨酸残基或其他磷酸化氨基酸的肽，证明了该方法对含P-His肽的专属性。
Phosphorylation is the predominant posttranslational modification involved in regulating enzymatic activity and mediating signal transduction in prokaryotic and eukaryotic cells. Selective enrichment of phosphorylated peptides prior to mass spectrometric analysis facilitates identification of phosphorylated proteins, determination of specific phosphorylated residues, and characterization of the conditions under which phosphorylation occurs. Such protocols have been established for peptides containing residues that form phosphoesters, such as serine and threonine, using immobilized metal-ion affinity chromatography. Despite the importance of histidine phosphorylation in two-component signal transduction pathways, similar protocols for peptides containing phosphorylated histidine (P-His) residues have proven elusive, due to the instability of these modifications and the propensity of unphosphorylated histidines to interact with immobilized metals ions. We describe a method for the selective extraction of a P-His-containing peptide using immobilized copper(II) ions and disposable metal-chelating pipet tips (ZipTip(MC), Millipore). The method is contingent upon pH-dependent interactions between the phosphate group and immobilized copper(II) ions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with postsource decay confirms the identity and phosphorylation state of the extracted peptide. Peptides containing unphosphorylated histidine residues or other phosphorylated amino acids are not retained, demonstrating the specificity of the method for P-His-containing peptides.