title: Identification of microRNA targets by pulsed SILAC.
abstract: 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.
authors: Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-Universität München, Thalkirchner Strasse 36, 80337, Munich, Germany.
authors: Markus
authors: M
authors: Kaller
authors: Silke
authors: S
authors: Oeljeklaus
authors: Bettina
authors: B
authors: Warscheid
authors: Heiko
authors: H
authors: Hermeking
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D053000:Analytic Sample Preparation Methods; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008156:Luciferases / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000906:Antibodies; D035683:MicroRNAs; D008156:Luciferases; D014357:Trypsin