Tp. Wallis et al., Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% O-18 water, PROTEIN SCI, 10(11), 2001, pp. 2251-2271
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.