Nonprotein based enrichment method to analyze peptide cross-linking in protein complexes

Anal Chem. 2009 Sep 1;81(17):7149-59. doi: 10.1021/ac900360b.


Cross-linking analysis of protein complexes and structures by tandem mass spectrometry (MS/MS) has advantages in speed, sensitivity, specificity, and the capability of handling complicated protein assemblies. However, detection and accurate assignment of the cross-linked peptides are often challenging due to their low abundance and complicated fragmentation behavior in collision-induced dissociation (CID). To simplify the MS analysis and improve the signal-to-noise ratio of the cross-linked peptides, we developed a novel peptide enrichment strategy that utilizes a cross-linker with a cryptic thiol group and using beads modified with a photocleavable cross-linker. The functional cross-linkers were designed to react with the primary amino groups in proteins. Human serum albumin was used as a model protein to detect intra- and intermolecular cross-linkages. Use of this protein-free selective retrieval method eliminates the contamination that can result from avidin-biotin based retrieval systems and simplifies data analysis. These features may make the method suitable to investigate protein-protein interactions in biological samples.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Cross-Linking Reagents / chemistry*
  • Crystallography, X-Ray
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Peptides / analysis*
  • Peptides / chemistry
  • Photolysis
  • Proteins / analysis*
  • Proteins / chemistry
  • Serum Albumin / analysis*
  • Serum Albumin / chemistry
  • Sulfhydryl Compounds / chemistry*
  • Tandem Mass Spectrometry / economics
  • Tandem Mass Spectrometry / methods*


  • Cross-Linking Reagents
  • Peptides
  • Proteins
  • Serum Albumin
  • Sulfhydryl Compounds