Letter abstract


Nature Biotechnology 21, 921 - 926 (2003)
Published online: 20 July 2003 | doi:10.1038/nbt849

A proteomics approach to understanding protein ubiquitination

Junmin Peng1,4, Daniel Schwartz1,5, Joshua E Elias1,5, Carson C Thoreen1,2, Dongmei Cheng2, Gerald Marsischky3, Jeroen Roelofs1, Daniel Finley1 & Steven P Gygi1,2

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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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  1. 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 to: Steven P Gygi1,2 e-mail: steven_gygi@hms.harvard.edu



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