Single-label fluorescent derivatization of peptides

A simple method for single-label fluorescent derivatization of peptides through their inherent primary α-mamino groups is described. The method relies on using the amine-reactive probe, FITC, and a lower-than-normal derivatization-buffer pH that discriminates derivatization of ε-amino groups from α-amino groups, provided that the pKa of the α-amino group is sufficiently high for it to act as an efficient nucleophile. A kinetic study for the derivatization through each of the two amino groups was performed in order to identify the optimal derivatization-buffer pH for α-amino group selectivity. This was found to be pH 8.5. It was found that single-label derivatization by buffer pH control was successful for peptides with fewer than four lysine residues for every N-terminus.