Identification of an insulin fragment produced by an insulin degrading enzyme, neutral thiopeptidase

Identification by fast atom bombardment mass spectrometry of insulin fragments produced by insulin proteinase

“…The results obtained confirm and extend previous work with insulin-degrading proteinase Davies et al, 1986Davies et al, , 1988Varandani & Shroyer, 1987). Varandani & Shroyer (1987) identified a fragment which had the expected N-termini and (approximate) amino acid composition of A1-13 and B1-9 linked by an interchain disulphide bond.…”

“…Varandani & Shroyer (1987) identified a fragment which had the expected N-termini and (approximate) amino acid composition of A1-13 and B1-9 linked by an interchain disulphide bond. We did not identify B1-10, which was found by Davies et al (1988), possibly because the fragment was not present in high enough yield in the two digests which we examined.…”

“…Fraction 4 was found to correspond to the major degradation product as deduced from the results of nonmass-spectrometric experiments Davies et al, 1986;Varandani & Shroyer, 1987). The positive-ion mass spectrum shows signals at m/z 1004 and 1353, corresponding to Bl-9 and A 1-13, respectively (Fig.…”

“…The degradation of insulin in vitro by insulin proteinase produces at least seven fragments, which are not easy to resolve completely in a primary separation step, given the rather small amounts available (Davies et al, , 1987Varandani & Shroyer, 1987). Identification of the degradation products, which is complicated to some extent by the two-chain nature of the substrate (insulin), has been performed by three quite different approaches.…”

“…Identification of the degradation products, which is complicated to some extent by the two-chain nature of the substrate (insulin), has been performed by three quite different approaches. One approach involves a combination of N-terminal analysis and amino acid analysis of unlabelled insulin fragments (Duckworth et al, 1979;Varandani & Shroyer, 1987). A second approach involves the use of 3H-labelled insulins as substrates, and comparison with standards during separations involving combinations of electrophoretic and chromatographic systems with and without cleavage of disulphide bonds by performic acid oxidation Davies et al, 1986Davies et al, , 1987.…”