title: A proteomics approach to understanding protein ubiquitination.
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.
authors.affiliation: Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.forename: Daniel
authors.initials: D
authors.lastname: Schwartz
authors.forename: Joshua E
authors.initials: JE
authors.lastname: Elias
authors.forename: Carson C
authors.initials: CC
authors.lastname: Thoreen
authors.forename: Dongmei
authors.initials: D
authors.lastname: Cheng
authors.forename: Gerald
authors.initials: G
authors.lastname: Marsischky
authors.forename: Jeroen
authors.initials: J
authors.lastname: Roelofs
authors.forename: Daniel
authors.initials: D
authors.lastname: Finley
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
mesh_terms: D000595:Amino Acid Sequence; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin