Extensive Peptide Fractionation and y1 Ion-based Interference Detection Enable Accurate Quantification by Isobaric Labeling and Mass Spectrometry

Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from co-isolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection and post-MS computational interference correction. We pooled tandem mass tag (TMT) labeled E. coli peptides at 1 : 3 : 10 ratios, and added in ~20-fold more rat peptides as background, followed by the analysis of two dimensional liquid chromatography (LC)-MS/MS. The quantitative interference was systematic investigated and the interference in MS2 scans were estimated by the intensity of contaminated y1 product ions. An algorithm was thus developed to correct reporter ion ratios of tryptic peptides.