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A major goal in proteomics is to accurately measure changes in the relative abundance of large sets of proteins in complex biological systems. Isobaric mass tagging with ExacTag labels is a powerful platform for simultaneously analyzing relative protein levels from multiple complex samples. The technology provides a superior approach, combining high precision and reproducibility to accurately determine the relative expression level of individual proteins in a complex mixture. A bioinformatics approach has been developed to enable fast and accurate protein identification and quantification through analyzing ExacTag-labeled peptides from MS/MS spectra. This approach combines popular protein identification programs such as Mascot or Sequest with an internally developed quantification software package. The results of the protein identification are used to predict labeled peptides, including possible posttranslational modifications, which can be used for quantification. In parallel, each spectrum in the MS/MS data is scanned for ions coming from the mass tag labels. The package then searches the quantification results, matching the precursor mass to predicted peptides, and generates a list of quantified peptides for each protein.
This study focuses on accurately assessing the quantitative consistency of the labeling technology, without introducing the confounding issue of biological variability. Complex samples were prepared consisting of different defined ratios of human serum and Escherichia coli bacterial cellular lysate. The protein mixtures were then labeled with different cysteine-reactive tags in order to compare protein expression levels among the different samples all at once using tandem mass spectrometry. The measured differences in protein levels correlated well with the known input ratios of the two complex samples, reproducing changes in abundance of over 20-fold range. The described technology provides a powerful method for enhancing proteomic analysis by mass spectrometry.