Optimization and applications of CDAP labeling for the assignment of cysteines.

The aim of the study is to provide a methodology for assigning unpaired cysteine residues in proteins formulated in a variety of different conditions to identify structural heterogeneity as a potential cause for protein degradation. 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was employed for cyanylating free cysteines in proteins and peptides. Subsequent basic cleavage of the peptide bond at the N-terminal side of the cyanylated cysteines provided direct information about their location. CDAP was successfully employed to a wide variety of labeling conditions. CDAP was reactive between pH 2.0 and 8.0 with a maximum labeling efficiency at pH 5.0. Its reactivity was not affected by excipients, salt or denaturant. Storing CDAP in an organic solvent increased its intrinsic stability. It was demonstrated that CDAP can be employed as a thiol-directed probe to investigate structural heterogeneity of proteins by examining the accessibility of unpaired cysteine residues. CDAP is a unique cysteine-labeling reagent because it is reactive under acidic conditions. This provides an advantage over other sulfhydryl labeling reagents as it avoids potential thiol-disulfide exchange. Optimization of the cyanylation reaction allowed the utilization of CDAP as a thiol-directed probe to investigate accessibility of sulfhydryl groups in proteins under various formulation conditions to monitor structural heterogeneity.