In vivo quantitative proteome profiling: planning and evaluation of SILAC experiments.
Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex biological samples. Improved instrumentation, quantification strategies and data analysis tools now enable protein analysis on a genome-wide scale. Particularly, quantification based on stable isotope labeling with amino acids (SILAC) has emerged as a robust, reliable and simple method for accurate large-scale protein quantification. The spectrum of applications ranges from bacteria and eukaryotic cell culture systems to multicellular organisms. Here, we provide a step-by-step protocol on how to plan and perform large-scale quantitative proteome analysis using SILAC, from sample preparation to final data analysis.
Cell Signalling and Mass Spectrometry Group, Max Delbrueck Center for Molecular Medicine, Berlin, Germany.
Marieluise
M
Kirchner
Matthias
M
Selbach
D000596:Amino Acids; D000818:Animals; D056148:Chromatography, Reverse-Phase; D003627:Data Interpretation, Statistical; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007553:Isotope Labeling; D010449:Peptide Mapping; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012107:Research Design; D012984:Software; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D000596:Amino Acids; D020543:Proteome; D014357:Trypsin