Peptide biotinylation with amine-reactive esters: Differential side chain reactivity

N-hydroxysuccinimide (NHS) esters of biotin are reported to react specifically with amino groups of peptides and proteins. However, we have found that these reagents can readily acylate other functional groups in specific peptide sequences under relatively mild conditions. We have extended our inquiry of sequence-dependent acylation by evaluating the reactivity of a variety of commonly employed biotinylation reagents typically used for amino group modification. These included the p-nitrophenyl ester of biotin, NHS- esters of biotin containing aminohexanoic acid spacer arms, and a sulfonated NHS-biotin ester that contained a disulfide bond within its spacer. The decapeptide [D-Lys⁶]gonadotropin releasing hormone was employed as a model peptide. Reaction products were characterized by high-performance liquid chromatography, amino acid compositional analysis, reaction with hydroxylamine, and mass spectrometry. In addition to the O-acylation of Ser⁴ and Tyr⁵ in this peptide, we have also identified a novel biotinylation of the Arg⁸ side chain.