Collision-induced Dissociative Chemical Cross-Linking Reagents and Methodology: Applications to Protein Structural Characterization Using Tandem Mass Spectrometry Analysis

Anal Chem. 2006 Dec 1;78(23):8059-68. doi: 10.1021/ac0613840.


Chemical cross-linking combined with mass spectrometry is a viable approach to study the low-resolution structure of protein and protein complexes. However, unambiguous identification of the residues involved in a cross-link remains analytically challenging. To enable a more effective analysis across various MS platforms, we have developed a novel set of collision-induced dissociative cross-linking reagents and methodology for chemical cross-linking experiments using tandem mass spectrometry (CID-CXL-MS/MS). These reagents incorporate a single gas-phase cleavable bond within their linker region that can be selectively fragmented within the in-source region of the mass spectrometer, enabling independent MS/MS analysis for each peptide. Initial design concepts were characterized using a synthesized cross-linked peptide complex. Following verification and subsequent optimization of cross-linked peptide complex dissociation, our reagents were applied to homodimeric glutathione S-transferase and monomeric bovine serum albumin. Cross-linked residues identified by our CID-CXL-MS/MS method were in agreement with published crystal structures and previous cross-linking studies using conventional approaches. Common LC/MS/MS acquisition approaches such as data-dependent acquisition experiments using ion trap mass spectrometers and product ion spectral analysis using SEQUEST were shown to be compatible with our CID-CXL-MS/MS reagents, obviating the requirement for high resolution and high mass accuracy measurements to identify both intra- and interpeptide cross-links.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aspartic Acid / chemistry
  • Cross-Linking Reagents / chemistry*
  • Glutathione Transferase / chemistry*
  • Glutathione Transferase / genetics
  • Glutathione Transferase / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Proline / chemistry
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Schistosoma japonicum / genetics
  • Schistosoma japonicum / metabolism
  • Serum Albumin, Bovine / chemistry*
  • Serum Albumin, Bovine / genetics
  • Serum Albumin, Bovine / metabolism*
  • Tandem Mass Spectrometry / methods*


  • Cross-Linking Reagents
  • Recombinant Fusion Proteins
  • Serum Albumin, Bovine
  • Aspartic Acid
  • Proline
  • Glutathione Transferase