DISULFIDE BOND MAPPING OF PROTEINS BY CYANYLATION

对蛋白质结构的了解需要确定是哪一种

A knowledge of protein structure requires a determination of which cysteine is connected to which cysteine. We cyanylate and cleave the peptide bond at the N-terminus of free cysteine residues. From the known sequence, we can anticipate the mass of degraded peptides for a given disulfide bond connectivity. We presently use cyanodiaminopyridium (CDAP) tetrafluoroborate, which cyanylates the protein at pH 3 to avoid disulfide bond exchange. We have established conditions for partial reduction at low pH which also avoids disulfide bond exchange. We have used this approach with ribonuclease A, which gives four singly reduced isomers, each having a different disulfide bond reduced. These isoforms were cyanylated and cleaved for analysis by MALDI-MS allowing us to verify the pairing of the disulfide bonds. We are also exploring the applicability of alternate cyanylating reagents to provide us with some flexibility in the chemical approach to solving a structural problem. We have synthesized a new reagent, 2-thiocyanopyridine, which will be characterized for its potency in cyanylating model compounds such as ?-lactoglobulin and others.