Optimization of the cleavage reaction for cyanylated cysteinyl proteins for efficient and simplified mass mapping.

Peptide chains can be cleaved selectively on the N-terminal side of cysteine residues after cyanylation of sulfhydryl groups to form an amino-terminal peptide and a series of 2-iminothiazolidine-4-carboxyl peptides. This paper describes a systematic study that was carried out to elucidate the effects of peptide structure and reaction conditions on the kinetics of the reactions and the yields of cleavage products. Both cleavage and beta-elimination reactions under different conditions (pH 8.0, 9.0, and 12.0 and 1 M ammonia solution) were quantitatively evaluated using reversed-phase HPLC and matrix-assisted laser desorption/ionization-MS. Contrary to previous reports, our results showed that higher pH greatly accelerates both cleavage and beta-elimination reactions, while the relative yield of beta-elimination products does not increase for most of the peptides studied. Optimal results were obtained in 1 M ammonium hydroxide solution, in which cleavage is complete within an hour at ambient temperature. This improvement also minimizes side reactions otherwise associated with long hours of exposure to alkaline conditions, [original report called for 12 to 80 h of incubation in mildly alkaline (pH 8-9) buffer]. The yields of cleavage reactions depend primarily on the structure of amino acids on the N-terminal side of cyanylated cysteines; the Pro-Cys and Tyr-Cys bonds were resistant to cleavage, promoting beta-elimination as the main reaction. The improved cleavage conditions greatly simplify the analytical procedure, which has been successfully applied to the determination of cysteine status in spinach ferredoxin, ovalbumin, and rabbit muscle creatine phosphokinase.