MASS MAPPING TO SHOW DISULFIDE BOND CONNECTIVY IN PROTEINS W/ ADJACENT CYSTEINES

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Conventional approaches to disulfide bond mapping rely on proteases to cleave the peptide backbone between all cysteine residues. Because proteases cannot cleave between adjacent cysteines, proteins containing this structural feature are refractory to the conventional approach. We have demonstrated that our cyanylation methodology can cleave the adjacent cysteine residues in long R3 insulin-like growth factor (LR3IGF), an 83-residue protein containing six cysteines in the form of three disulfide bonds. Analysis of the cleavage products of two singly reduced isoforms of LR3IGF by LC-MS using electrospray ionization verified that cleavage between Cys60 and Cys61 had been achieved. The results of this study also proved that the disulfide connectivity in LR3IGF-1 is homologous to that in insulin-like growth factor (IGF-1).