title: High Sensitivity Crosslink Detection Coupled With Integrative Structure Modeling in the Mass Spec Studio.
abstract: 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.
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Sarpe
authors.affiliation: §Department of Chemistry.
authors.forename: Atefeh
authors.initials: A
authors.lastname: Rafiei
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology.
authors.forename: Morgan
authors.initials: M
authors.lastname: Hepburn
authors.affiliation: ¶Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ostan
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, ¶Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Schryvers
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology, §Department of Chemistry, dschriem@ucalgary.ca.
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012984:Software; D013552:Swine; D053719:Tandem Mass Spectrometry; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*; D033882:Transferrin-Binding Protein B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D010455:Peptides; D014168:Transferrin; D033882:Transferrin-Binding Protein B