In vitro modification of human centromere protein CENP-C fragments by small ubiquitin-like modifier (SUMO) protein: definitive identification of the modification sites by tandem mass spectrometry analysis of the isopeptides

J Biol Chem. 2004 Sep 17;279(38):39653-62. doi: 10.1074/jbc.M405637200. Epub 2004 Jul 22.


Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif psiKXE/D, where psi represents a large hydrophobic amino acid, and X represents any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chromosomal Proteins, Non-Histone / chemistry*
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Consensus Sequence
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Peptide Mapping
  • Protein Processing, Post-Translational*
  • Repressor Proteins / metabolism*
  • SUMO-1 Protein / metabolism*
  • Small Ubiquitin-Related Modifier Proteins
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Ubiquitin-Conjugating Enzymes / metabolism


  • Chromosomal Proteins, Non-Histone
  • Peptide Fragments
  • Repressor Proteins
  • SUMO-1 Protein
  • SUMO2 protein, human
  • Small Ubiquitin-Related Modifier Proteins
  • centromere protein C
  • Ubiquitin-Conjugating Enzymes
  • ubiquitin-conjugating enzyme UBC9