Kaller Markus, Oeljeklaus Silke, Warscheid Bettina, Hermeking Heiko
Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-Universität München, Thalkirchner Strasse 36, 80337, Munich, Germany.
Methods Mol Biol. 2014;1188:327-49. doi: 10.1007/978-1-4939-1142-4_23.
Pulsed stable isotope labeling by amino acids in cell culture (pulsed SILAC or pSILAC) allows to monitor and quantify the de novo synthesis of proteins in an unbiased fashion on a proteome-wide scale. The high applicability of this metabolic labeling technique has been demonstrated for the identification of posttranscriptional changes in gene expression on the proteome level, in particular those caused by microRNAs. The application of pSILAC allows the selective quantification of newly synthesized proteins and thus the detection of differences in protein translation. This is of particular interest in the case of microRNA-mediated regulations, which characteristically cause rather modest decreases in protein amounts that may be difficult to detect by other proteomic methods. Here, we describe a detailed protocol for using pSILAC to track miRNA-mediated changes in protein expression, using the p53-induced miR-34a microRNA as a prototypic example of microRNA-mediated regulations.
细胞培养中氨基酸的脉冲稳定同位素标记（脉冲SILAC或pSILAC）能够在蛋白质组范围内以无偏差的方式监测和定量蛋白质的从头合成。这种代谢标记技术在蛋白质组水平上鉴定基因表达的转录后变化，特别是由微小RNA引起的变化方面，已显示出高度的适用性。pSILAC的应用允许对新合成的蛋白质进行选择性定量，从而检测蛋白质翻译中的差异。在微小RNA介导的调控情况下，这一点尤为重要，微小RNA介导的调控通常会导致蛋白质含量适度下降，而这可能难以通过其他蛋白质组学方法检测到。在这里，我们以p53诱导的miR-34a微小RNA作为微小RNA介导调控的典型例子，描述了使用pSILAC追踪微小RNA介导的蛋白质表达变化的详细方案。
