Nested Arg-specific bifunctional crosslinkers for MS-based structural analysis of proteins and protein assemblies

Anal Chim Acta. 2008 Oct 3;627(1):117-28. doi: 10.1016/j.aca.2008.05.074. Epub 2008 Jun 5.


The combination of chemical probing and high-resolution mass spectrometry constitutes a powerful alternative for the structural elucidation of biomolecules possessing unfavorable size, solubility, and flexibility. We have developed nested Arg-specific bifunctional crosslinkers to obtain complementary information to typical Cys- and Lys-specific reagents available on the market. The structures of 1,4-phenyl-diglyoxal (PDG) and 4,4'-biphenyl-diglyoxal (BDG) include two identical 1,2-dicarbonyl functions capable of reacting with the guanido group of Arg residues in proteins, as well as the base-pairing face of guanine in nucleic acids. The reactive functions are separated by modular spacers consisting of one or two benzene rings, which confer greater rigidity to the crosslinker structure than it is afforded by typical aliphatic spacers. Analysis by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has shown that the probes provide both mono- and bifunctional products with model protein substrates, which are stabilized by the formation of diester derivatives in the presence of borate buffer. The identification of crosslinked sites was accomplished by employing complementary proteolytic procedures and peptide mapping by ESI-FTICR. The results showed excellent correlation with the solvent accessibility and structural context of susceptible residues, and highlighted the significance of possible dynamic effects in determining the outcome of crosslinking reactions. The application of nested reagents with different spacing has provided a new tool for experimentally recognizing flexible regions that may be involved in prominent dynamics in solution. The development of new bifunctional crosslinkers with diverse target specificity and different bridging spans is expected to facilitate the structure elucidation of progressively larger biomolecular assemblies by increasing the number and diversity of spatial constraints available for triangulating the position of crosslinked structures in the three dimensions.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arginine / metabolism*
  • Cattle
  • Chemistry Techniques, Analytical / instrumentation*
  • Cross-Linking Reagents / chemistry*
  • Cross-Linking Reagents / metabolism*
  • Humans
  • Models, Molecular
  • Phenylglyoxal / analogs & derivatives
  • Phenylglyoxal / chemistry
  • Phenylglyoxal / metabolism
  • Protein Conformation
  • Proteins / chemistry*
  • Proteins / metabolism*
  • Spectrometry, Mass, Electrospray Ionization
  • Substrate Specificity


  • Cross-Linking Reagents
  • Proteins
  • 1,4-phenyldiglyoxal
  • Arginine
  • Phenylglyoxal