Use of Proteinase K Nonspecific Digestion for Selective and Comprehensive Identification of Interpeptide Cross-links: Application to Prion Proteins

• Published in: Molecular & cellular proteomics 2012-07, Vol.11 (7), p.M111.013524-1-M111.013524-13
• Main Authors: Petrotchenko, Evgeniy V., Serpa, Jason J., Hardie, Darryl B., Berjanskii, Mark, Suriyamongkol, Bow P., Wishart, David S., Borchers, Christoph H.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biotin; Chromatography, Affinity; Cricetinae; Cross-Linking Reagents; Endopeptidase K - metabolism; Escherichia coli; Mesocricetus; Models, Molecular; Molecular Sequence Data; Peptides - analysis; Peptides - chemistry; Peptides - genetics; Prions - analysis; Prions - chemistry; Prions - genetics; Proteolysis; Recombinant Proteins - analysis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M111.013524

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 (PrPC) 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 PrPC 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 Lys185–Lys220 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.