Impact of Peptide Modifications on the Isobaric Tags for Relative and Absolute Quantitation Method Accuracy

摘要

ABSTRACT: In this study, the impact of amino acid modificationsnon the accuracy of the iTRAQ (isobaric tags for relative andnabsolute quantitation) method was evaluated. MCF-7 breastncancer cells, cultured in the presence of 17β-estradiol andntamoxifen, were used as a model system. The cells were labelednand analyzed by reversed-phase liquid chromatography andnpulsed Q dissociation ion trap tandem mass spectrometryndetection. Database searching was performed by using variousncombinations of amino acid modification allowances, i.e, LysTyr/Cys and amino terminal iTRAQ labeling, Lys methylation,nacetylation and carbamylation, and Cys/Met oxidation. Othernthan the intended Lys/amino terminal iTRAQ labeling, suchnmodifications occur as a result of either enzymatic or sample preparation related reactions and are typically ignored in quantitationnanalysis to minimize the rate of false-positive peptide identifications. The study revealed that the modifications with the greatestnimpact on protein identification and quantitation pertain to Lys and Tyr amino acid residues, that by enabling suchmodifications thennumber and type of identified proteins will change (by up to 10%), and that the rate of false-positive protein identifications can benmaintained below an upper threshold of 5% if appropriate data filtering conditions are used. In addition, the interference of possiblenposttranslational modifications (i.e., phosphorylation) with iTRAQ quantitation was examined.