SILAC for the study of mammalian cell lines and yeast protein complexes.
Through crucial advancements in quantitative mass spectrometry (MS), proteomics has evolved from taking mere "snapshots" of proteomes to thoroughly studying dynamic changes in entire proteomes and characterizing intricate protein-protein interaction or signaling networks. Thus, quantitative MS-based proteomics offers the unique potential to place proteins into their functional context and, moreover, to improve our understanding of the molecular processes involved in the development, survival, or pathology of cells and organisms. Among the vast variety of techniques developed for the accurate quantification of proteins via MS, stable isotope labeling by amino acids in cell culture (SILAC) arguably represents the most elegant method. In this chapter, we provide a detailed protocol for the establishment of SILAC for mammalian cell culture systems. In addition, to exemplify the high versatility of SILAC for addressing different biological questions, we describe the successful "pairing" of SILAC with conventional affinity purification (AP)-MS approaches allowing for accurately characterizing protein complexes.
Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
Heike
H
Piechura
Silke
S
Oeljeklaus
Bettina
B
Warscheid
D000596:Amino Acids; D000818:Animals; D018929:Cell Culture Techniques; D002457:Cell Extracts / Q000737:chemistry / Q000302:isolation & purification; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification; D014357:Trypsin / Q000737:chemistry
D000596:Amino Acids; D002457:Cell Extracts; D046912:Multiprotein Complexes; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin