Improved method for identification and determination of .epsilon.-(.gamma.-glutamyl)lysine cross-link in protein using proteolytic digestion and derivatization with phenyl isothiocyanate followed by high-performance liquid chromatography separation

The cross-link was liberated by exhaustive proteolytic digestion and derivatized with PITC. The derivatives were resolved by HPLC. The present method enables one to determine simultaneously protein-constituting amino acids and ∈-(γ-glucyl)lysine and thereby facilitates the monitoring of progress of proteolytic digestion. The PTC-∈-(γ-glutamyl)lysine peak was collected and confirmed by conversion to a stable PTH form followed by HCl hydrolysis