Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces

J Am Chem Soc. 2006 Aug 16;128(32):10362-3. doi: 10.1021/ja0614159.


Chemical cross-linking followed by identification of the cross-linked residues by mass spectrometry provides structural information on protein interaction surfaces. Nevertheless, accurate analysis of the digested, cross-linked proteins is often challenging. Herein, we describe a novel strategy that relies on the use of affinity-tagged cross-linkers and isotope coding on the cross-linker-modified species. Incorporation of O16 or O18 during the hydrolysis of the cross-linkers results in a characteristic "doublet" for the undesired products of a half-cross-linking reaction. Therefore, genuine cross-linked peptides are readily distinguished for further structural analysis. This strategy permits a sensitive and facile analysis on a dimeric protease inhibitor, ecotin, showing general applicability to other protein assemblies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels*
  • Cross-Linking Reagents / chemistry*
  • Crystallography, X-Ray
  • Escherichia coli Proteins / chemistry
  • Isotope Labeling / methods*
  • Models, Molecular
  • Molecular Structure
  • Periplasmic Proteins / chemistry
  • Proteins / chemistry*
  • Surface Properties


  • Affinity Labels
  • Cross-Linking Reagents
  • Eco protein, E coli
  • Escherichia coli Proteins
  • Periplasmic Proteins
  • Proteins