α-Amidated Peptides: Approaches for Analysis

Abstract

alpha-Amidation is a terminal modification in peptide biosynthesis that can itself be rate-limiting in the overall production of bioactive alpha-amidated peptides. More than half of the known neural and endocrine peptides are alpha-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 alpha-amidated peptides. The principle analytical method used to quantify alpha-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) the performance of the RIA itself. Techniques are also described for the identification and verification of alpha-amidated peptides. Lastly, in vivo models used for studying the biology of alpha-amidation are discussed.