iTRAQ analysis

What is iTRAQ?

iTRAQ is a stable isotope method for relative protein quantitation using mass spectrometry. It is a primary amine specific label that will label all peptide N-termini. Up to 4 labels are availble allowing for a more dynamic approach including time course analyses and biological replicates. Due to the large number of peptides produced, we perform SCX chromatography on iTRAQ samples prior to LC-MS/MS or LC-MALDI TOF/TOF MS/MS.

Advantages

• all tryptic peptides are labelled resulting in increased confidence and higher quality data
• up to 4 lables can be used for multiplexing experiments
• improved ms/ms fragmentation results in better confidence id's
• post translational modifications can be detected

Disadvantages

• more mass spec time is required becasue of the increased number of peptides
• samples must be prepared according to strict guidelines

Link to Applied Biosystems website

Instruments and Software used for iTRAQ analysis

-- An Applied Biosystems API QStar Pulsar mass spectrometer is used to acquire iTRAQ data in our lab. The QStar mass spectrometer is interfaced with an LC Packings Ultimate Nanoflow HPLC System.

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