title: Proteomic identification and analysis of K63-linked ubiquitin conjugates.
abstract: Post-translational modification of proteins by covalent attachment of ubiquitin or a polyubiquitin chain is involved in myriad of processes in eukaryotic cells. The particular outcome of ubiquitination is directed by the length of the ubiquitin conjugate and its linkage composition. Among seven possible isopeptide linkage sites in ubiquitin, K48 and K63 occur most commonly and act as distinct cellular signals. Strategies are reported here for analysis of linkage sites and complexity of K63-linked polyubiquitin chains, based on rapid chemical proteolysis at aspartate residues combined with immunoprecipitation and mass spectrometry. Rapid chemical proteolysis at aspartate residues results in K63-linked peptides with truncated branches, which enable identification and characterization of stretches of consecutive K63 linkages on generally available instruments. A characteristic cleavage pattern and a characteristic fragmentation pattern allow recognition of K63 oligomers in proteolytic mixtures. Engineered K63-linked polyubiquitin chains of defined lengths were used to evaluate and demonstrate the method. In-gel microwave-supported acid hydrolysis was used to observe peptides specific to K63-linked ubiquitin dimers and trimers. Acid hydrolysis in solution, used in conjunction with linkage-specific immunoprecipitation, allowed more complex K63-linked branches to be characterized. Finally, a substrate protein, UbcH5b, was conjugated to monoubiquitin and to polyubiquitin chains containing only K63 linkages, and the sites of conjugation and chain lengths were characterized.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States.
authors.forename: Joe
authors.initials: J
authors.lastname: Cannon
authors.forename: Mark
authors.initials: M
authors.lastname: Nakasone
authors.forename: David
authors.initials: D
authors.lastname: Fushman
authors.forename: Catherine
authors.initials: C
authors.lastname: Fenselau
mesh_terms: D000595:Amino Acid Sequence; D047468:Immunoprecipitation; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; D008239:Lysine