A proteomics approach to understanding protein ubiquitination.
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.
Peng
Junmin
J
Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, Massachusetts 02115, USA.
Schwartz
Daniel
D
Elias
Joshua E
JE
Thoreen
Carson C
CC
Cheng
Dongmei
D
Marsischky
Gerald
G
Roelofs
Jeroen
J
Finley
Daniel
D
Gygi
Steven P
SP
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*
D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin