Strategies based on sum of peak intensities may better reflect protein expression than peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform

Abstract

In the field of quantitative proteomics, the Isobaric Tags for Relative & Absolute Quantitation (iTRAQ) technology has demonstrated efficacy for proteome monitoring despite its lack of a consensus for data handling. In this 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 = 0.2962, p = 0.0099 **) with an average bias of 0.08747 ± 0.5004 and 95% Limits of Agreement from - 0.8932 to 1.068. We proposed the Mascot-jTRAQx-CiR-C strategy as a simple yet powerful data processing adjunct to the iTRAQ technology.

Serum Metabolic Profiling Analysis of Chronic Gastritis and Gastric Cancer by Untargeted Metabolomics

“…AB SCIEX ® TripleTOF 5600+ Plus ultra-performance liquid chromatography-tandem mass spectrometer (UHPLC-Q-TOF/MS) was used to acquire the MS/MS spectra on information-dependent basis during the LC/MS experiment. In this mode, acquisition software (Analyst TF1.7 software) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria ( 20 ). The mobile phase containing 0.1% formic acid was composed of water (A) and acetonitrile (B) at a flow rate of 0.3 ml/min.…”