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.
Mesh Terms:
Amino Acid Sequence, Gene Expression Regulation, Fungal, Molecular Sequence Data, Protein Interaction Mapping, Proteome, Proteomics, Saccharomyces cerevisiae Proteins, Sequence Alignment, Sequence Analysis, Protein, Sequence Homology, Amino Acid, Ubiquitin
Nat. Biotechnol.
Date: Aug. 01, 2003
60865
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