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Mol Cell Proteomics 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, S-E, Jaffe, JD, Carr, SA
JournalMol Cell Proteomics
Volume11
Issue6
PagesM111.014423
Date Published2012 Jun
ISSN1535-9484
KeywordsEpidermal Growth Factor, HeLa Cells, Humans, Isotope Labeling, Peptide Fragments, Phosphoproteins, Proteome, Proteomics, Regression Analysis, Tandem Mass Spectrometry
Abstract

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, nonisobaric 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 threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although 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 cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of 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 nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.

URLhttp://www.mcponline.org/cgi/pmidlookup?view=long&pmid=22210691
DOI10.1074/mcp.M111.014423
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/22210691?dopt=Abstract

Alternate JournalMol. Cell Proteomics
PubMed ID22210691
PubMed Central IDPMC3433912
Grant ListU24 CA160034 / CA / NCI NIH HHS / United States
R01HL096738 / HL / NHLBI NIH HHS / United States
R01 HL096738 / HL / NHLBI NIH HHS / United States
HHSN268201000033C / / PHS HHS / United States
HHSN268201000033C / HL / NHLBI NIH HHS / United States
U24CA160034 / CA / NCI NIH HHS / United States