Sum of peak intensities outperforms 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 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.

CIL-ExPMRM: An Ultrasensitive Chemical Isotope Labeling Assisted Pseudo-MRM Platform to Accelerate Exposomic Suspect Screening

“…The peak heights for these compounds could often be obtained directly from MS software without requiring manual correction. The applicability of intensity in the quantification was based on the condition that a Gaussian distributed chromatographic peak shape exists across different samples, 43 which is usually true for the case for most DnsCl-or MPEA-modified compounds. Here, peak height, instead of peak area, was used for relative quantification even for extremely low abundance compounds after derivatization, which was another advantage of the CIL strategy.…”

Multi-Evaluating Strategy for Siji-kangbingdu Mixture: Chemical Profiling, Fingerprint Characterization, and Quantitative Analysis

“…This technology can provide large data capacity and the deep detection of chemical compounds in herbal medicines. Additionally, with a compound library, we can easily search for chemical compounds in samples with satisfactory confidence levels [17,18]. The fingerprint similarity analysis method has been accepted worldwide to reflect the internal quality of herbal medicines by showing their holistic and fuzzy characteristics, which is consistent with TCM methodology [19,20,21].…”