title: Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins.
abstract: Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including a Lys(185)-Lys(220) cross-link, which is unique to the PrPβ and thus may be indicative of the conformational change involved in the formation of prion protein oligomers.
authors.affiliation: University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, Victoria, British Columbia V8Z 7X8, Canada.
authors.forename: Evgeniy V
authors.initials: EV
authors.lastname: Petrotchenko
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Serpa
authors.forename: Darryl B
authors.initials: DB
authors.lastname: Hardie
authors.forename: Mark
authors.initials: M
authors.lastname: Berjanskii
authors.forename: Bow P
authors.initials: BP
authors.lastname: Suriyamongkol
authors.forename: David S
authors.initials: DS
authors.lastname: Wishart
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin; D002846:Chromatography, Affinity; D006224:Cricetinae; D003432:Cross-Linking Reagents; D019286:Endopeptidase K / Q000378:metabolism*; D004926:Escherichia coli; D008647:Mesocricetus; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D011328:Prions / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D059748:Proteolysis; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides; D011328:Prions; D011994:Recombinant Proteins; D001710:Biotin; D019286:Endopeptidase K