Antibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W

Nat Commun. 2021 Jul 29;12(1):4608. doi: 10.1038/s41467-021-24669-6.


The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / chemistry*
  • Antibodies / immunology
  • Cells, Cultured
  • Crystallography, X-Ray / methods
  • Humans
  • Mass Spectrometry / methods
  • Peptides / chemistry*
  • Protein Binding
  • Proteomics / methods
  • Rabbits
  • Ubiquitin-Conjugating Enzymes / chemistry
  • Ubiquitin-Conjugating Enzymes / immunology
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitinated Proteins / metabolism*
  • Ubiquitination


  • Antibodies
  • Peptides
  • Ubiquitinated Proteins
  • UBE2W protein, human
  • Ubiquitin-Conjugating Enzymes