isobaric Tags for Relative and Absolute Quantitation (iTRAQ)

Biological samples (4, 6 or 10) for comparison can be differentially labelled using an amine specific label that is attached to each peptide after trypsin digestion. The first step is to accurately measure the samples protein concentration to ensure that the same amount of protein is labelled in each sample. Once each sample is quantified, it is digested and differentially labelled before being combined into either 4, 6 or 10 plex experiments and then fractionated to reduce complexity prior to further analysis. Fractionation is usually by High pH reverse phase with each fraction being run by 1D nano LC-MS/MS on a LTQ Orbitrap Velos Pro ms system. The ms data for each fraction is then combined prior to database searching and quantitation by either Proteome Discoverer using the Mascot search engine or MaxQuant, which use the MS/MS data to produce both relative quantitation and identification of proteins from each sample.