Analysis of iodinated peptides by LC-DAD/ESI ion trap mass spectrometry

The analysis of iodinated peptides resulting from chloramine-T (CAT), Iodo-Beads®, Iodo-Gen® and lactoperoxidase iodination reactions in the preparation of nanomole quantities 125I and 123I labelled tracers is described. Seven different model peptides were evaluated, varying in molecular weight from 294 (LY-dipeptide) to 2518 (obestatin containing 23 amino acid residues). Two different RP-C18 columns were used, each with a different gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting components of the reaction mixtures, while UV (DAD) served quantitative purposes. Non-, mono-, di-, tri- and tetra-iodinated peptides (respectively NIP, MIP, DIP, 3IP and 4IP) eluted in that order and were well separated from each other. An empirical model was derived. The applicability of this approach was demonstrated by the analysis of different reaction mixtures.