Phosphate-specific fluorescence labeling of pepsin by BO-IMI

Pepsin (3.6 nmol) was detected by the following three-step procedure: (i) reaction with a 20-fold molar excess of BO-IMI (a fluorophore containing a reactive imidazole group) in the presence of a 150-fold molar excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2% sodium dodecyl sulfate; (ii) gel filtration (spin column) to remove most of the residual BO-IMI; and (iii) capillary electrophoresis with laser-induced fluorescence detection. For the latter step, 8.5 x 10(-7) of the original sample was injected. BO-IMI/EDC targets phosphomonoesters and does not label albumin (prior knowledge). Progressive dephosphorylation of pepsin with acid phosphatase reduced its labeling with BO-IMI. Thus, the BO-IMI, as intended, labels the phosphate group on pepsin. Such BO-IMI labeling should be useful in general for studying phosphoproteins and phosphopeptides.