Determination of epsilon (gamma-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation.

A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the epsilon (gamma-glutamyl)lysine isopeptide bond, which is usually formed by protein-crosslinking transglutaminases between polypeptide chains. The procedure is based on the separation and quantitation of epsilon (gamma-glutamyl)lysine isodipeptide following exhaustive proteolytic digestion of the crosslinked peptide. It involves preliminary separation steps on a cation exchanger resin and a silica HPLC column, precolumn derivatization with phenylisothiocyanate, and reversed-phase high-pressure liquid chromatographic separation on a C18 column. The derivatized isodipeptide gave a linear concentration-response relationship, with a detection limit of 10 pmol/mg of protein. The combination of the preliminary separation steps and the sensitive detection system permits the determination of the epsilon (gamma-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.