Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling

Anal Chem. 2002 Sep 1;74(17):4417-22. doi: 10.1021/ac0257492.


A new method is presented to screen proteolytic mass maps of cross-linked protein complexes for the presence of cross-linked peptides and for the verification of proposed structures. On the basis of the incorporation of 18O from isotopically enriched water into the C-termini of proteolytic peptides, cross-linked peptides are readily distinguished in mass spectra by a characteristic 8 amu shift. This is due to the incorporation of two 18O atoms in each C-terminus, so that normal and surface-labeled peptides shift 4 amu and cross-linked peptides containing two C-termini will shift 8 amu compared with their unlabeled counterparts. The method is fast, sensitive, and reliable and can be combined with any available cross-linking reagent and a wide range of proteolytic agents. As proof of principle, we successfully applied the method to a complex of two DNA repair proteins (Rad18-Rad6) and identified the interaction domain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cross-Linking Reagents
  • DNA-Binding Proteins / analysis
  • DNA-Binding Proteins / metabolism
  • Ligases / analysis
  • Ligases / metabolism
  • Mass Spectrometry / methods
  • Mass Spectrometry / standards*
  • Oxygen Isotopes
  • Peptide Mapping / methods
  • Peptide Mapping / standards*
  • Protein Binding
  • Proteins / analysis*
  • Proteins / metabolism
  • Saccharomyces cerevisiae Proteins*
  • Ubiquitin-Conjugating Enzymes


  • Cross-Linking Reagents
  • DNA-Binding Proteins
  • Oxygen Isotopes
  • Proteins
  • RAD18 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin-Conjugating Enzymes
  • Ligases