A knowledge of protein structure requires a determination of which cysteine is connected to which cysteine. The classical approach requires isolating the cystinyl peptides after enzymatic digestion, if possible. Our approach requires less material than the classical enzymatic approach, and minimizes exposure to disulfide exchange. Our goal is to provide a simplified technique involving little or no separation by HPLC. We use nitrothiocyanobenzoic acid (NTCB) which chemically cleaves the peptide bond at the N-terminus of Cys. From the known sequence, one can anticipate the mass of degraded peptides for a given disulfide bond connectivity. By considering all possible connectivities, one can anticipate the patterns of peaks from which to recognize the pairing of cysteines in a given disulfide bond. Our strategy requires that in a protein containing multiple disulfide bonds that we analyze a form of the analyte in which only one of them has been reduced in a given structure. We have established conditions for partial reduction at low pH which also avoids disulfide bond exchange. We have used this approach with ribonuclease which gives 4 singly reduced isomers, each having a different disulfide bond pair. These isoforms were cyanylated and cleaved for analysis by MALDI-MS allowing us to verify the pairing of the disulfide bonds.
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