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

Analytical Biochemistry
E Tarcsa, L Fesus

Abstract

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.

References

Oct 1, 1988·Analytical Biochemistry·S A Cohen, D J Strydom
Apr 1, 1971·Proceedings of the National Academy of Sciences of the United States of America·J J PisanoY Nagai
Aug 1, 1980·Proceedings of the National Academy of Sciences of the United States of America·M L FinkJ E Folk
Dec 7, 1984·Journal of Chromatography·B A BidlingmeyerT L Tarvin

Citations

Mar 4, 1991·Biochimica Et Biophysica Acta·J HarsfalviA G Loewy
Jun 17, 2005·Analytical Biochemistry·Zoltan NemesL Fesus
Jul 5, 2005·Analytical Biochemistry·András MádiL Fesus
Oct 12, 1999·Journal of Chromatography. B, Biomedical Sciences and Applications·M L Miller, G V Johnson
Oct 6, 1997·American Journal of Respiratory Cell and Molecular Biology·J C SchittnyD Aeschlimann

Related Concepts

Phenylisothiocyanate
Blood Platelets
High Pressure Liquid Chromatography Procedure
Cross-Linking Reagents
Hydrolysis
Ion Exchange
Osmolality
Statistical Sensitivity
Thiocyanates
Reproducibility of Results

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