Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing

J Proteome Res. 2006 Sep;5(9):2270-82. doi: 10.1021/pr060154z.

Abstract

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein-protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer software. This method is specifically tailored toward the rapid analysis of low microgram amounts of proteins or multimeric protein complexes cross-linked with nonlabeled and deuterium-labeled bis-NHS ester cross-linking reagents (both commercially available and readily synthesized). Through labeling with [18O]water solvent and LC-MALDI analysis, the method further allows the possible distinction between Type 0 and Type 1 or Type 2 modified peptides (monolinks and looplinks or cross-links), although such a distinction is more readily made from analysis of tandem mass spectrometry data. When applied to the bacterial Colicin E7 DNAse/Im7 heterodimeric protein complex, 23 cross-links were identified including six intersubunit cross-links, all between residues that are close in space when examined in the context of the X-ray structure of the heterodimer. In addition, cross-links were successfully identified in five single subunit proteins, beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A, establishing the generality of the approach.

Publication types

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

MeSH terms

  • Chromatography, Liquid
  • Computational Biology / methods
  • Cross-Linking Reagents / chemistry*
  • Protein Conformation*
  • Proteins / chemistry*
  • Proteomics / methods*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Cross-Linking Reagents
  • Proteins