Impact of peptide modifications on the isobaric tags for relative and absolute quantitation method accuracy.

In this study, the impact of amino acid modifications on the accuracy of the iTRAQ (isobaric tags for relative and absolute quantitation) method was evaluated. MCF-7 breast cancer cells, cultured in the presence of 17β-estradiol and tamoxifen, were used as a model system. The cells were labeled and analyzed by reversed-phase liquid chromatography and pulsed Q dissociation ion trap tandem mass spectrometry detection. Database searching was performed by using various combinations of amino acid modification allowances, i.e, Lys/Tyr/Cys and amino terminal iTRAQ labeling, Lys methylation, acetylation and carbamylation, and Cys/Met oxidation. Other than the intended Lys/amino terminal iTRAQ labeling, such modifications occur as a result of either enzymatic or sample preparation related reactions and are typically ignored in quantitation analysis to minimize the rate of false-positive peptide identifications. The study revealed that the modifications with the greatest impact on protein identification and quantitation pertain to Lys and Tyr amino acid residues, that by enabling such modifications the number and type of identified proteins will change (by up to 10%), and that the rate of false-positive protein identifications can be maintained below an upper threshold of 5% if appropriate data filtering conditions are used. In addition, the interference of possible posttranslational modifications (i.e., phosphorylation) with iTRAQ quantitation was examined.