Systems biology "on-the-fly": SILAC-based quantitative proteomics and RNAi approach in Drosophila melanogaster.
Stable isotope labeling with amino acids in cell culture (SILAC) has become increasingly popular as a quantitative proteomics (qProteomics) method. In combination with high-resolution mass spectrometry (MS) and new efficient algorithms for the analysis of quantitative MS data, SILAC has proven to be a potent tool for the in-depth characterization of functional states. QProteomics extends transcriptomics analysis in providing comprehensive and unbiased protein expression profiles. In this chapter, we describe the use of SILAC procedure in combination with RNA interference (RNAi) to characterize loss-of-function phenotypes, an example to illustrate how qProteomics can address many of the systems-wide approaches previously restricted to the mRNA level. Furthermore, by explaining the adaptation of SILAC to a novel cellular model, the Drosophila melanogaster Schneider cells SL2, we aim to offer an example enabling the readers to apply the same strategy to any other cell culture, specific for their need.
Cuomo
Alessandro
A
Department of Molecular Oncology, European Institute of Oncology, Milano, Italy.
Bonaldi
Tiziana
T
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D010641:Phenotype; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism
D000596:Amino Acids; D012330:RNA, Double-Stranded