Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% O-18 water

Determination of the disulfide-bond arrangement of a protein by characteriz ation of disulfide-linked peptides in proteolytic digests may be complicate d by resistance of the protein to specific proteases, disulfide interchange , and/or production of extremely complex mixtures by less specific proteoly sis. In this study, mass spectrometry has been used to show that incorporat ion of O-18 into peptides during peptic digestion of disulfide-linked prote ins in 50% O-18 water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-l inked peptides even in complex mixtures, without the need for reference dig ests in 100% O-16 water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted l aser desorption/ionization-time of flight (MALDI-TOF) and electrospray ioni zation (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of th e chains did not contain the C terminus of the protein, This latter class o f peptide, and single-chain peptides containing an intrachain disulfide bon d, could be identified and characterized by mass shifts produced by reducti on. Reduction also served to confirm other assignments. Isotope profiling o f peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatog raphy (SEC) of the digests. Applicability of these procedures to analysis o f a more complex disulfide-bond arrangement was shown with the hemagglutini n/neuraminidase of Newcastle disease virus.