iTRAQ Labeling is Superior to mTRAQ for Quantitative Global Proteomics and Phosphoproteomics

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. 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. Stable isotope labeling techniques have become very popular in recent years to perform quantitative mass spectrometry experiments with high precision and accuracy. In contrast to label-free approaches, multiplexed isotopically labeled samples can be simultaneously analyzed resulting in increased reproducibility and accuracy for quantification of peptides and proteins from different biological states. Isotopic labeling strategies can be grouped into two major categories: isobaric labels and nonisobaric labels. In the former category are iTRAQ 1The abbreviations used are:iTRAQisobaric tags for relative and absolute quantificationmTRAQmass differential tags for relative and absolute quantificationTMTtandem mass tagsSILACstable isotope labeling by amino acids in cell cultureCIDcollision-induced dissociationHCDhigher-energy collisional dissociationPIPprecursor isolation purityMRMmultiple reaction monitoringSCXstrong cation exchangeEGFepidermal growth factorEGFRepidermal growth factor receptor. 1The abbreviations used are:iTRAQisobaric tags for relative and absolute quantificationmTRAQmass differential tags for relative and absolute quantificationTMTtandem mass tagsSILACstable isotope labeling by amino acids in cell cultureCIDcollision-induced dissociationHCDhigher-energy collisional dissociationPIPprecursor isolation purityMRMmultiple reaction monitoringSCXstrong cation exchangeEGFepidermal growth factorEGFRepidermal growth factor receptor. (isobaric tags for relative and absolute quantification (1Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar)) and TMT (tandem mass tags (2Thompson A. Schäfer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Johnstone R. Mohammed A.K. Hamon C. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 75: 1895-1904Crossref PubMed Scopus (1709) Google Scholar)) mass tags. In the nonisobaric labeling category are methods such as mTRAQ (mass differential tags for relative and absolute quantification), stable isotope labeling by amino acids in cell culture (SILAC (3Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar)), and reductive dimethylation (4Boersema P.J. Raijmakers R. Lemeer S. Mohammed S. Heck A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.Nat. Protoc. 2009; 4: 484-494Crossref PubMed Scopus (1055) Google Scholar). Isobaric labeling techniques allow relative quantification of peptides based on ratios of low m/z reporter ions produced by fragmentation of the precursor ion, whereas nonisobaric labeling yields precursors with different masses that can be directly quantified from MS1 intensity. iTRAQ and mTRAQ reagents provide a great opportunity to directly compare capabilities of reporter and precursor ion quantification since both labels have identical chemical structures and differ only in their composition and number of 13C, 15N, and 18O atoms. In fact, iTRAQ-117 and mTRAQ-Δ4 are identical mass tags with a total mass of 145 Da (Fig. 1A). To achieve 4-plex quantification capabilities for iTRAQ labels, the composition of stable isotopes is arranged in a way to obtain the reporter ion/balancing group and (1Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar). nonisobaric mTRAQ labels were by or to the mTRAQ-Δ4 resulting in and Both iTRAQ and mTRAQ reagents are as to primary labeling of isobaric tags for relative and absolute quantification mass differential tags for relative and absolute quantification mass tags stable isotope labeling by amino acids in cell culture collisional precursor isolation reaction cation growth factor growth factor receptor. isobaric tags for relative and absolute quantification mass differential tags for relative and absolute quantification mass tags stable isotope labeling by amino acids in cell culture collisional precursor isolation reaction cation growth factor growth factor receptor. of an iTRAQ labeling strategy is additive effect on precursor samples are resulting in increased However, iTRAQ ratios have to be to nonregulated background peptides are and cofragmented in the same isolation of the peptide of and to the reporter ions in accuracy and precision in iTRAQ Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google M. J. C. I. iTRAQ in simple and complex the and the 2009; PubMed Scopus Google F. T. G. M. M. for reproducibility and of mass spectrometry PubMed Scopus Google Scholar). most peptides in an are ratios multiplexed ratios in the to be toward to the of mTRAQ labels to accurate quantification of proteins in iTRAQ experiments with such as reaction reaction of peptides absolute quantification of of a in and PubMed Scopus Google Scholar). Although iTRAQ has used in mTRAQ has only in a number of reaction of peptides absolute quantification of of a in and PubMed Scopus Google Scholar). In this we the and of iTRAQ and mTRAQ labeling for analysis of protein and expression We growth factor HeLa as a for comparative of iTRAQ and mTRAQ as both in the Blagoev B. F. B. C. P. Mann M. in and in signaling Full Text Full Text PDF PubMed Scopus Google as as the J. 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Our results indicate that in of are to redundant of the same peptide in different isotopically labeled in or reductive dimethylation In to of MS2 be be into and for quantification key proteins in the EGFR signaling network were quantified with both techniques, indicating that of protein the of quantification for both iTRAQ- and However, the of protein found in the were detected and quantified only in iTRAQ indicating a to of of iTRAQ ratios because of nonregulated background peptides has in a number of accuracy and precision in iTRAQ Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google M. J. C. I. iTRAQ in simple and complex the and the 2009; PubMed Scopus Google F. T. G. M. M. for reproducibility and of mass spectrometry PubMed Scopus Google Scholar). and defined a for iTRAQ quantification and reproducibility of quantification results for protein high that to high fragmentation. is very to we have used in this in the We observed only correlation of high with quantification accuracy of peptides ratios into complex is because of the that the of reporter ions in iTRAQ are to the of the precursor ions from ions of can iTRAQ reporter ions that over two orders of in intensity. that very low background ions can significantly to reporter ion cofragmented with the precursor ion the MS2 is be used as an accurate to for quantified iTRAQ ratios in and has that iTRAQ ratio compression a an and results in a linear observed and ratios of proteins accuracy and precision in iTRAQ Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). 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However, in two recent peptide fragmentation have to iTRAQ reporter ion that of that are on precursors multiplexed quantification with isobaric PubMed Scopus Google or on high m/z ions R. ratio in isobaric multiplexed quantitative proteomics.Nat. PubMed Scopus Google Scholar). of be to iTRAQ quantification accuracy and to iTRAQ ratio compression In we show that iTRAQ reagents are to mTRAQ reagents to of novel regulated in and iTRAQ quantification less and reproducibility than quantification with accuracy to compression of iTRAQ ratios not the of regulated phosphopeptides and with