Ratio accuracy evaluation and correction using tandem mass tags

The multiplexing capabilities of isobaric mass tag based protein quantification, such as Tandem Mass Tags (TMT) or Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) have dramatically increased the scope of Mass Spectrometry (MS) based proteomics studies. Not only does the technology allow for the simultaneous quantification of multiple samples in a single MS injection, but its seamless compatibility with extensive sample pre-fractionation methods allow for comprehensive study of complex proteomes. However, reporter ion based quantification has often been criticized for limited quantification accuracy due to interference from co-eluting peptides and peptide fragments. In this study, we investigate the extent of this problem and propose an effective and easy-to-implement remedy relying on spiking a 6-protein calibration mixture to the samples subject to relative quantification. Our ratio adjustment approach was evaluated on a large scale TMT 10-plex dataset derived from cancer cell lines with chromosome instability. Furthermore, we analyzed a complex 2-proteome artificial sample mixture and investigated the precision of TMT and precursor ion intensity based Label Free Quantification. Comparing the two methods we observed that the isobaric tag based quantification workflow, due to its superior precision, was able to confirm more and smaller protein abundance changes, at a fixed False Positive Rate.