Isobaric tag-based quantification such as iTRAQ and TMT is a promising approach to mass spectrometry-based quantification in proteomics as it provides wide proteome coverage with greatly increased experimental throughput. However, it is known to suffer from inaccurate quantification and identification of a target peptide due to cofragmentation of multiple peptides, which likely leads to under-estimation of differentially expressed peptides (DEPs). A simple method of filtering out cofragmented spectra with less than 100% precursor isolation purity (PIP) would decrease the coverage of iTRAQ/TMT experiments. In order to estimate the impact of cofragmentation on quantification and identification of iTRAQ-labeled peptide samples, we generated multiplexed spectra with varying degrees of PIP by mixing the two MS/MS spectra of 100% PIP obtained in global proteome profiling experiments on gastric tumor-normal tissue pair proteomes labeled by 4-plex iTRAQ. Despite cofragmentation, the simulation experiments showed that more than 99% of multiplexed spectra with PIP greater than 80% were correctly identified by three different database search engines-MODa, MS-GF+, and Proteome Discoverer. Using the multiplexed spectra that have been correctly identified, we estimated the effect of cofragmentation on peptide quantification. In 74% of the multiplexed spectra, however, the cancer-to-normal expression ratio was compressed, and a fair number of spectra showed the "ratio inflation" phenomenon. On the basis of the estimated distribution of distortions on quantification, we were able to calculate cutoff values for DEP detection from cofragmented spectra, which were corrected according to a specific PIP and probability of type I (or type II) error. When we applied these corrected cutoff values to real cofragmented spectra with PIP larger than or equal to 70%, we were able to identify reliable DEPs by removing about 25% of DEPs, which are highly likely to be false positives. Our experimental results provide useful insight into the effect of cofragmentation on isobaric tag-based quantification methods. The simulation procedure as well as the corrected cutoff calculation method could be adopted for quantifying the effect of cofragmentation and reducing false positives (or false negatives) in the DEP identification with general quantification experiments based on isobaric labeling techniques.
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