iTRAQ is a technique that utilizes a multiplexed isobaric chemical tagging reagent which allows multiplexing of two to eight protein samples and produces identical MS/MS sequencing ions for all eight versions of the same derivatized tryptic peptide. This greatly facilitates peptide identification due to the resulting higher intensities of the parent and fragment ions. Quantitation is achieved by comparison of the peak areas and resultant peak ratios for either four MS/MS reporter ions, which range from 114 to 117 Da or eight MS/MS reporter ions, which range from 113-119 and 121 Da. Mass spectrometric analysis is done on an AB QSTAR Elite mass spectrometer with Protein Pilot (AB) software quantitation.
- samples must be prepared in a buffer containing no primary amines
- Peptides are both identified and quantified with the same experiment by multi dimensional LC and MS/MS
- Quantitation is done at the MS/MS spectral level, and typically 400 or more identified proteins from a crude cellular or tissue extract are quantified.
- Amino Acid Analysis (AAA) is used to accurately determine the protein concentration of each sample
- Recommended sample amount is 25 µg (8 Plex) to 100µg (4 plex)