High Sensitivity Crosslink Detection Coupled With Integrative Structure Modeling in the Mass Spec Studio

Mol Cell Proteomics. 2016 Sep;15(9):3071-80. doi: 10.1074/mcp.O116.058685. Epub 2016 Jul 13.


The Mass Spec Studio package was designed to support the extraction of hydrogen-deuterium exchange and covalent labeling data for a range of mass spectrometry (MS)-based workflows, to integrate with restraint-driven protein modeling activities. In this report, we present an extension of the underlying Studio framework and provide a plug-in for crosslink (XL) detection. To accommodate flexibility in XL methods and applications, while maintaining efficient data processing, the plug-in employs a peptide library reduction strategy via a presearch of the tandem-MS data. We demonstrate that prescoring linear unmodified peptide tags using a probabilistic approach substantially reduces search space by requiring both crosslinked peptides to generate sparse data attributable to their linear forms. The method demonstrates highly sensitive crosslink peptide identification with a low false positive rate. Integration with a Haddock plug-in provides a resource that can combine multiple sources of data for protein modeling activities. We generated a structural model of porcine transferrin bound to TbpB, a membrane-bound receptor essential for iron acquisition in Actinobacillus pleuropneumoniae Using mutational data and crosslinking restraints, we confirm the mechanism by which TbpB recognizes the iron-loaded form of transferrin, and note the requirement for disparate sources of restraint data for accurate model construction. The software plugin is freely available at www.msstudio.ca.

MeSH terms

  • Actinobacillus pleuropneumoniae / chemistry
  • Actinobacillus pleuropneumoniae / genetics
  • Actinobacillus pleuropneumoniae / metabolism*
  • Animals
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cross-Linking Reagents / chemistry*
  • Models, Molecular
  • Mutation
  • Peptides / analysis*
  • Peptides / chemistry
  • Protein Binding
  • Protein Conformation
  • Software
  • Swine
  • Tandem Mass Spectrometry
  • Transferrin / chemistry
  • Transferrin / metabolism*
  • Transferrin-Binding Protein B / chemistry
  • Transferrin-Binding Protein B / genetics
  • Transferrin-Binding Protein B / metabolism*


  • Bacterial Proteins
  • Cross-Linking Reagents
  • Peptides
  • Transferrin
  • Transferrin-Binding Protein B