Structural characterization of pyrrolic cross-links in collagen using a biotinylated Ehrlich's reagent

The structures of pyrrolic forms of cross-links in collagen have been confi rmed by reacting collagen peptides with a biotinylated Ehrlich's reagent. T his reagent was synthesized by converting the cyano group of N-methyl-N-cya noethyl-4-aminobenzaldehyde to a carboxylic acid, followed by conjugation w ith biotin pentylamine. Derivatization of peptides from bone collagen both stabilized the pyrroles and facilitated selective isolation of the pyrrole- containing peptides using a monomeric avidin column. Reactivity of the biot inylated reagent with collagen peptides was similar to that of the standard Ehrlich reagent, but heat denaturation of the tissue before enzyme digesti on resulted in the loss of about 50% of the pyrrole cross-links. Identifica tion of a series of peptides by mass spectrometry confirmed the presence of derivatized pyrrole structures combined with between 1 and 16 amino acid r esidues. Almost all of the pyrrole-containing peptides appeared to be deriv ed from N-terminal telopeptide sequences, and the nonhydroxylated (lysine-d erived) form predominated over pyrrole cross-links derived from helical hyd roxylysine.