Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

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Abstract

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.

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Figure 1: Structure of the ICAT reagent.
Figure 2: The ICAT strategy for quantifying differential protein expression.
Figure 3: Isotope-coded affinity tag quantitative analysis of a protein from the mixture in Table 1.
Figure 4: The ICAT sequence identification of the peptide quantified in Figure 3 as derived from glyceraldehyde-3-phosphate dehydrogenase.
Figure 5: Isotope-coded affinity tag analysis of alcohol dehydrogenase isozymes in yeast.

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Acknowledgements

This work was supported in part by the NSF Science and Technology Center for Molecular Biotechnology; NIH grants RR11823, T32HG00035, and HD-02274; a grant from the Merck Genome Research Institute; University of Washington Royalty Research Fund grant; and a fellowship to B.R. from the Swiss National Science Foundation. We thank D.R. Goodlett and J. Watts for critical reading of the manuscript.

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Correspondence to Ruedi Aebersold.

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