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Molecular & cellular proteomics : MCP DOI:10.1074/mcp.M111.014423

iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics.

Publication TypeJournal Article
Year of Publication2012
AuthorsMertins, P, Udeshi, ND, Clauser, KR, Mani, DR, Patel, J, Ong, SE, Jaffe, JD, Carr, SA
JournalMolecular & cellular proteomics : MCP
Date Published2011/12/30

Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ or TMT allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, non-isobaric labeling with mTRAQ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly 3-fold more phosphopeptides (12,129 vs. 4,448) and nearly 2-fold more proteins (2,699 vs. 1,597) than mTRAQ labeling. While most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are co-fragmented in the same precursor isolation window, dampening observed ratios toward unity. However, due to tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of non-regulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.