α-Amidated Peptides: Approaches for Analysis

Abstract

α-Amidation is a terminal modification in peptide biosynthesis that can itself be rate limiting in the overall production of bioactive α-amidated peptides. More than half of the known neural and endocrine peptides are α-amidated and in most cases this structural feature is essential for receptor recognition, signal transduction, and thus biologic function. This chapter describes methods for developing and using analytical tools to study the biology of α-amidated peptides. The principal analytical method used to quantify α-amidated peptides is the radioimmunoassay (RIA). Detailed protocols are provided for (1) primary antibody production and characterization; (2) radiolabeling of RIA peptides; (3) sample preparation; and (4) performance of the RIA itself. Techniques are also described for the identification and verification of α-amidated peptides. Lastly, in vivo models used for studying the biology of α-amidation are discussed.