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Biomolecular Research Institute, Parkville VIC 3052, Australia
Reprint requests to: Dr. Jeffrey J. Gorman, CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville VIC 3052, Australia; e-mail: jeff.gorman{at}hsn.csiro.au; fax: 61-3-9662-7101.
Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of 18O into peptides during peptic digestion of disulfide-linked proteins in 50% 18O water resulted in isotope patterns and increases in average masses that facilitated ideication and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% 16O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be ideied and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.
Abbreviations: DLP, disulfide-linked peptide • HPLC, high performance liquid chromatography • ESI, electrospray ionization • MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight • MS, mass spectrometry • LC-MS, liquid chromatography-mass spectrometry • RNaseA, bovine pancreatic ribonuclease A • SEC, size exclusion chromatography • RP-HPLC, reverse phase HPLC • NDV HN, Newcastle disease virus hemagglutinin/neuraminidase
Keywords: Mass spectrometry; disulfide-linked peptides; peptic digestion; oxygen isotope; isotope profiles
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