An aptly positioned azido group in the spacer of a protein cross-linker for facile mapping of lysines in close proximity

Chembiochem. 2007 Jul 23;8(11):1281-92. doi: 10.1002/cbic.200700150.

Abstract

Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine-specific cross-linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. One pathway leads to cleavage of the cross-linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP-induced reaction products. These can be isolated by reversed-phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross-link-derived reaction products can be isolated and identified indicates that the mapping of cross-links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Azides / chemistry*
  • Chromatography, High Pressure Liquid
  • Cross-Linking Reagents / chemistry*
  • Cytochromes c / chemistry*
  • Horses
  • Lysine / chemistry*
  • Molecular Sequence Data
  • Molecular Structure
  • Peptides / chemistry
  • Phosphines / chemistry
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Azides
  • Cross-Linking Reagents
  • Peptides
  • Phosphines
  • tris(2-carboxyethyl)phosphine
  • Cytochromes c
  • Lysine