iTRAQ Multiplex Protein Quantitation

by LC-MS/MS analysis and database searching

Differential protein expression profiling can be obtained using multiplex tagging reagents and LC MS peptide analysis. The tryptic peptides from each biological sample are labelled with one of the tagging reagents, such as iTRAQ or di-methyl reagents, the peptides are combined, separated and purified by SCX or HILIC chromatography. The peptides are then analysed by LC MS/MS on a QTOF or Orbitrap mass spectrometer. The MS/MS data are used for database searching and peptide identification, and the differential labels are used for relative quantification of each peptide.

Advantages:

Thousands of peptides identified and quantified in one single analysis
Large dynamic range, high and low abundant proteins are identified
High diversity of proteins observed, including acidic, basic, high Mw, and hydrophobic membrane proteins

The iTRAQ Concept

: The concept of iTRAQ Reagent chemistry. Each sample is labeled with one of the 4 iTRAQ reagents and then pooled prior to peptide fractionation and LC MS/MS analysis. Peptides are identified by sequence specific fragment ions, and relatively quantified by the 114, 115, 116, 117 reporter ions (ref: Applied Biosystems Application note on iTRAQ Reagents).