Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs)

Mol Cell Proteomics. 2016 Apr;15(4):1381-96. doi: 10.1074/mcp.o115.051839.


Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Humans
  • Mass Spectrometry / methods
  • Protein Binding
  • Proteomics / methods*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Ubiquitin / metabolism*
  • Ubiquitinated Proteins / chemistry
  • Ubiquitinated Proteins / isolation & purification*


  • Saccharomyces cerevisiae Proteins
  • Ubiquitin
  • Ubiquitinated Proteins