iTRAQ-Based Shotgun Proteomics Approach for Relative Protein Quantification

Shotgun proteomics has a key role in quantitative estimation of proteins from biological systems under different conditions, which is crucial in the understanding of their functional roles. Isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry is based on pre-labeling of peptides with mass tags which allows the multiplex analysis of up to eight proteomes simultaneously. We describe here a detailed protocol for sample preparation and iTRAQ 4-plex labeling for relative quantification of multiple samples from human and plant tissues. We also present two strategies for peptide fractionation after the iTRAQ labeling protocol.