Methogo Ruth Menque, Dufresne-Martin Geneviève, Leclerc Patrice, Leduc Richard, Klarskov Klaus
Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
J Proteome Res. 2005 Nov-Dec;4(6):2216-24. doi: 10.1021/pr050014+.
The combined use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry has become a powerful and widely used tool in proteome studies. Following separation by electrophoresis, proteins can be transferred to an inert support such as polyvinylidene fluoride (PVDF) or nitrocellulose (NC) for the visualization of individual or specific classes of proteins by immunochemical detection methods. We developed a method that allows the mass spectrometric analysis of peptides derived from proteins detected by Western blotting on PVDF. Proteolysis buffer containing either dimethyl formamide (DMF) or Triton X-100 to recover peptides amenable to mass spectrometry was investigated. Although either one can be used, the buffer containing DMF required less sample handling prior to mass spectrometry. The approach was tested using commercially available proteins and serine-phosphorylated proteins from an HEK-293 nuclear extract.
十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）与质谱联用已成为蛋白质组学研究中一种强大且广泛应用的工具。通过电泳分离后，蛋白质可转移至诸如聚偏二氟乙烯（PVDF）或硝酸纤维素（NC）等惰性支持物上，以便通过免疫化学检测方法对单个或特定类别的蛋白质进行可视化分析。我们开发了一种方法，可对通过蛋白质印迹法在PVDF上检测到的蛋白质衍生的肽段进行质谱分析。研究了含有二甲基甲酰胺（DMF）或 Triton X-100 的蛋白酶解缓冲液，以回收适合质谱分析的肽段。虽然二者均可使用，但含有 DMF 的缓冲液在质谱分析前所需的样品处理较少。使用市售蛋白质和来自 HEK-293 细胞核提取物的丝氨酸磷酸化蛋白质对该方法进行了测试。
