Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid

CBT-Cys click condensation reaction has a high second-order reaction rate constant and has found wide applicability in recent years. However, its reaction mechanism has not been experimentally validated and its application for identifying bioactive N-terminal Cys peptides in real clinical samples has not been reported. Herein, firstly, by employing induced nanoelectrospray ionization-mass spectrometry (InESI-MS) and a home-built micro-reactor, we successfully intercepted and structurally characterized the crucial intermediate in this click reaction for the first time. With the intermediate, the proposed mechanism of this reaction was corroborated. Moreover, we also applied this MS setup to monitor the reaction in real time and obtained the second-order reaction rate constants of this reaction at different pH values. After mechanistic study, we applied this click reaction for identifying bioactive N-terminal cysteine peptides in amniotic fluid (AF). Eight unique N-terminal Cys peptides in AF, three of which are located in the functional domain regions of their corresponding proteins, were identified with a false positive rate less than 1%. One of the three peptides was found able to inhibit the growth of uterine endometrial cancer HEC-1-B cells but not the endometrial normal cells via a typical apoptotic pathway. With its mechanism satisfactorily elucidated, the kinetic parameters obtained, as well as its application for fishing bioactive N-terminal Cys peptides from vast complex clinical samples, we anticipate that this CBT-Cys click reaction could be applied more widely for the facile isolation, site-specific identification, and quantification of N-terminal Cys-containing peptides in complex biological samples.