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.