Proteomic identification and analysis of K63-linked ubiquitin conjugates

Joe Cannon, Mark Nakasone, David Fushman, Catherine Fenselau

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)10121-8
Number of pages8
JournalAnalytical Chemistry
Issue number22
Publication statusPublished - 20 Nov 2012


  • Amino Acid Sequence
  • Immunoprecipitation
  • Lysine
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Secondary
  • Proteomics/methods
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Ubiquitin/chemistry
  • Ubiquitination


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