To unequivocally confirm the covalent binding of the carbamate warhead to hBChEi.e., the carbamoylation of catalytic Ser198, we conducted a mass spectrometry study. Since the bound carbamate moiety could be hydrolysed under pepsinolytic conditions that are typically used for sample preparation for MS, whole protein mass spectrometry analysis using recombinant hBChE produced in E. coli was undertaken. The absence of glycosylation in a protein from prokaryotic expression system was needed to produce a uniform multicharged protein spectrum. In both control and carbamoylated samples, the protein eluted at 7.2 min under our experimental conditions and the deconvolution of spectra resulted in one major species for each sample.