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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics
Philipp Mertins1, Namrata D Udeshi, Karl R Clauser
1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Molecular & Cellular Proteomics : MCP
|January 3, 2012
Summary
Isobaric tags for relative and absolute quantitation (iTRAQ) identified more phosphopeptides and proteins than multiplexed quantitative peptide assay (mTRAQ). Despite iTRAQ
Area of Science:
- Proteomics
- Quantitative Mass Spectrometry
- Chemical Biology
Background:
- Stable isotope labeling is crucial for quantitative mass spectrometry.
- Isobaric labeling (e.g., iTRAQ) uses reporter ions for relative quantification.
- Non-isobaric labeling (e.g., mTRAQ) allows direct quantification in MS1 spectra.
Purpose of the Study:
- To compare the efficacy of iTRAQ and mTRAQ labeling for peptide and phosphopeptide quantification.
- To evaluate the impact of labeling strategies on protein and phosphopeptide identification.
- To assess the accuracy and variability of quantification methods.
Main Methods:
- Quantitative mass spectrometry analysis of EGF-stimulated HeLa cell peptides and phosphopeptides.
- Direct comparison of iTRAQ and mTRAQ labeling strategies.
- Analysis of precursor ion intensities, MS2 scanning events, and ratio distributions.
Main Results:
- iTRAQ identified significantly more phosphopeptides (nearly threefold) and proteins (nearly twofold) than mTRAQ.
- iTRAQ labeling showed an additive effect on precursor intensities; mTRAQ led to more redundant MS2 scans.
- While iTRAQ accuracy was affected by cofragmentation, its ratio distributions were tighter, yielding similar significant regulation percentages.
- A linear correlation between iTRAQ and mTRAQ ratios suggests potential for iTRAQ ratio correction.
Conclusions:
- iTRAQ labeling offers superior depth in phosphopeptide and protein quantification compared to mTRAQ.
- Both methods have distinct advantages and limitations impacting quantification accuracy and efficiency.
- Further research can explore correcting iTRAQ ratios for improved accuracy.
