Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform

In the field of quantitative proteomics, the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology has demonstrated efficacy for proteome monitoring despite its lack of a consensus for data handling. In the present study, after peptide and protein identification, we compared the widespread quantitation method based on the calculation of MS/MS reporter ion peaks areas ratios (ProteinPilot) to the alternative method based on the calculation of ratios of the sum of peak intensities (jTRAQx [Quant]) and we processed output data with the in-house Customizable iTRAQ Ratios Calculator (CiR-C) algorithm. Quantitation based on peak area ratios displayed no significant linear correlation with Western blot quantitation. In contrast, quantitation based on the sum of peak intensities displayed a significant linear association with Western blot quantitation (non-zero slope; Pearson correlation coefficient test, r = 0.296, P=0.010**) with an average bias of 0.087 ± 0.500 and 95% Limits of Agreement from −0.893 to 1.068. We proposed the Mascot-jTRAQx-CiR-C strategy as a simple yet powerful data processing adjunct to the iTRAQ technology.