Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: crystallography, activity profiling, and protease identification

Methods Enzymol. 2005:399:120-31. doi: 10.1016/S0076-6879(05)99008-3.


Isopeptidases that specifically remove ubiquitin or ubiquitin-like molecules from polypeptide adducts are emerging as key regulatory enzymes in a multitude of biochemical pathways. We have developed a set of tools that covalently target the active site of ubiquitin or ubiquitin-like deconjugating enzymes. We have used epitope-tagged ubiquitin and ubiquitin-like derivatives in immunoprecipitation assays to identify active proteases by mass spectrometry (MS/MS). The epitope tag confers the ability to conduct an immunoblot-based profiling assay for active isopeptidases in cell extracts. We have applied a ubiquitin-based probe in the structural analysis of the ubiquitin hydrolase UCH-L3 in its ligand-bound state. We describe the use of these electrophilic derivatives of ubiquitin and ubiquitin-like molecules in the identification, activity profiling, and structural analysis of these proteases. These tools can be used to rapidly profile activity of multiple Ub/UBL-specific proteases in parallel in cell extracts. We also show that in vitro these probes can be conjugated onto parts of the Ub/UBL conjugating machinery.

MeSH terms

  • Crystallography
  • Humans
  • Immunohistochemistry
  • Peptide Hydrolases / chemistry
  • Peptide Hydrolases / metabolism*
  • Proteomics*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Tandem Mass Spectrometry
  • Ubiquitin / chemistry
  • Ubiquitin / metabolism*


  • Recombinant Proteins
  • Ubiquitin
  • Peptide Hydrolases