Protein Profiling of the Brain: Proteomics of Isolated Tissues and Cells

A relatively new proteomic technology platform known as stable isotope labeling of amino acids in cell culture (SILAC) has been developed to record quantitative changes in in vitro experimental systems. This method allows for monitoring of changes due to various experimental conditions and also dynamics of changes in time or, e.g., during administration of drugs, etc. Furthermore, SILAC technology has advanced in such a way to include the analyses of protein turnover in both dividing and nondividing cells, a technology now known as pulse SILAC (pSILAC). The ability to profile proteomes of isolated cells or tissues of the brain opens new experimental opportunities in neuroscience. The focus of this protocol is to provide a step-by-step outline including brain tissue collection and isolation, sample preparation for iTRAQ and pSILAC labeling of primary cells, fractionation of proteins and in-gel tryptic digest, mass spectrometry-based identification and quantitation of peptides, and validation of differentially expressed proteins using Western blot.