Study of primary amines for nucleophilic cleavage of cyanylated cystinyl proteins in disulfide mass mapping methodology

In a study of primary (methyl to butyl) amines as nucleophiles for cyano-induced cleavage of cysteinyl proteins, methylamine was found to be superior to ammonia for cyanylation (CN)-based disulfide mass mapping methodology. Reaction conditions such as nucleophile concentration, temperature, and reaction time were systematically studied using ribonuclease A as a model protein. The CN-induced cleavage products were monitored using reverse-phase chromatography and matrix-assisted laser desorption ionization mass spectrometry. Results showed that low temperature, short reaction time, and high nucleophile concentration optimize the cleavage reaction and minimize side reactions. These conditions shorten the analysis time and substantially improve the yield of cleavage products. Further, the concurrent use of homologous nucleophiles (e.g., ammonia and methylamine) facilitates recognition and identification of cleavage products.