Abstract
There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.
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Acknowledgements
We thank T. Yao and R. Cohen for sharing ubiquitin proteins and J. Rush and M. Comb for phosphopeptide synthesis. We also thank A. Goldberg, R. Cohen, D. Moazed, S. Gerber and L. Licklider for encouraging discussions. This work was supported in part by National Institutes of Health grants HG00041 (S.P.G.), GM67945 (S.P.G.), GM43601 (D.F.), Giovani-Armenise Harvard Foundation (S.P.G.), Jane Coffin Childs Memorial Fund for Medical Research (J.P.) and a European Molecular Biology Organization long-term fellowship (J.R.).
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Peng, J., Schwartz, D., Elias, J. et al. A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21, 921–926 (2003). https://doi.org/10.1038/nbt849
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DOI: https://doi.org/10.1038/nbt849
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