Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE

Methods Enzymol. 2019:618:135-147. doi: 10.1016/bs.mie.2018.12.032. Epub 2019 Feb 1.


Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).

Keywords: Polyubiquitin chain; Substrate identification; TR-TUBE; Ubiquitin ligase; Ubiquitin-binding protein; Ubiquitination.

MeSH terms

  • Animals
  • Deubiquitinating Enzymes / metabolism
  • Enzyme Assays / methods
  • Humans
  • Polyubiquitin / metabolism
  • Substrate Specificity
  • Trypsin / metabolism
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitinated Proteins / metabolism*
  • Ubiquitination


  • Ubiquitin
  • Ubiquitinated Proteins
  • Polyubiquitin
  • Ubiquitin-Protein Ligases
  • Deubiquitinating Enzymes
  • Trypsin