Nature Biotechnology
21, 921 - 926 (2003)
Published online: 20 July 2003; | doi:10.1038/nbt849
A proteomics approach to understanding protein ubiquitinationJunmin Peng1, 4, Daniel Schwartz1, 5, Joshua E Elias1, 5, Carson C Thoreen1, 2, Dongmei Cheng2, Gerald Marsischky3, Jeroen Roelofs1, Daniel Finley1
& Steven P Gygi1, 21
Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA. 2
Taplin Biological Mass Spectrometry Facility, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA. 3
Department of Biochemistry and Molecular Pharmacology, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA. 4
Present address: Department of Human Genetics, Center for Neurodegenerative Disease, Emory University, 615 Michael Street, Atlanta, GA 30322, USA. 5
These authors contributed equally to this work.
Correspondence should be addressed to Steven P Gygi steven_gygi@hms.harvard.eduThere 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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