Determination of ε{lunate}(γ-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation

Edit Tarcsa, L. Fésüs

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41 Citations (Scopus)

Abstract

A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the ε{lunate}(γ-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 ε{lunate}(γ-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 ε{lunate}(γ-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.

Original languageEnglish
Pages (from-to)135-140
Number of pages6
JournalAnalytical Biochemistry
Volume186
Issue number1
DOIs
Publication statusPublished - 1990

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Lysine
Pressure
Liquids
Cation Exchange Resins
Peptides
Proteins
Transglutaminases
Silicon Dioxide
Limit of Detection
Digestion
Biological systems
High Pressure Liquid Chromatography
Crosslinking
Tissue
phenylisothiocyanate

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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title = "Determination of ε{lunate}(γ-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation",
abstract = "A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the ε{lunate}(γ-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 ε{lunate}(γ-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 ε{lunate}(γ-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.",
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N2 - A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the ε{lunate}(γ-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 ε{lunate}(γ-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 ε{lunate}(γ-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.

AB - A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the ε{lunate}(γ-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 ε{lunate}(γ-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 ε{lunate}(γ-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.

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