Stage 1. Breaking Disulphide Bonds: In protein two cysteine amino acids are linked by a disulphide linkage. The disulphide linkage interfere with the complete sequencing procedure as it doesn’t allow the release of cleaved amino acid from the peptide chain. There are two approaches two disrupt the disulphide linkage in a protein sequence (Figure 38.2). In first approach, protein is oxidized with a performic acid to produce two cysteic acid residues. In another approach, protein is reduced by dithiothreitol (DTT) or β-mercaptoethanol (β-me) to form two cysteine followed by treatment with iodoacetate to form carboxymethyl-cysteine. Formation of carboxymethyl-cysteine stops the re-formation of disulphide bond.

Figure 38.2: Disruption of disulphide bond by different approaches.