Abstract
Among a variety of global quantification strategies utilized in mass spectrometry (MS)-based proteomics, isobaric tags for relative and absolute quantitation (iTRAQ) are an attractive option for examining the relative amounts of proteins in different samples. The inherent complexity of mammalian proteomes and the diversity of protein physicochemical properties mean that complete proteome coverage is still unlikely from a single analytical method. Numerous options exist for reducing protein sample complexity and resolving digested peptides prior to MS analysis. Indeed, the reliability and efficiency of protein identification and quantitation from an iTRAQ workflow strongly depend on sample preparation upstream of MS. Here we describe our methods for: (1) total protein extraction from immortalized cells; (2) subcellular fractionation of murine tissue; (3) protein sample desalting, digestion, and iTRAQ labeling; (4) peptide separation by strong cation-exchange high-performance liquid chromatography; and (5) peptide separation by isoelectric focusing.