Stasyk Taras, Morandell Sandra, Bakry Rania, Feuerstein Isabel, Huck Christian W, Stecher Guenther, Bonn Guenther K, Huber Lukas A
Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria.
Electrophoresis. 2005 Jul;26(14):2850-4. doi: 10.1002/elps.200500026.
Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.
在这里，我们将标准的二维差异凝胶电泳（DIGE）方案与随后用磷特异性荧光Pro-Q Diamond染料对凝胶进行后染色相结合。这两种蛋白质荧光检测方法的结合使得在二维凝胶中能够定量检测磷酸化蛋白质。我们在一个功能蛋白质组学实验中建立了这个方案。乳腺上皮细胞（EpH4）在培养中受到表皮生长因子（EGF）的刺激，亚细胞分级分离后制备内体组分，并在内体上成功检测到磷酸化蛋白质。例如，通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）鉴定了Endo A细胞角蛋白，它是一种磷酸化蛋白质，也是由MAPK信号诱导的分化标志物。通过这个方案，蛋白质组和磷酸化蛋白质组分析实验的所有步骤都得到了显著简化和加速，在定量的特异性、灵敏度和准确性方面充分利用了这两种方法。
