The end product of transglutaminase crosslinking: simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] and lysine by HPLC-MS3

Transglutaminases catalyze the formation of Nepsilon-(gamma-glutamyl) isodipeptide crosslinks between proteins. These enzymes are thought to participate in a number of diseases, including neurological disease and cancer. A method associating liquid chromatography and multiple stage mass spectrometry...

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Bibliographic Details
Published inAnalytical biochemistry Vol. 384; no. 2; pp. 296 - 304
Main Authors Hoffner, G, van der Rest, G, Dansette, P M, Djian, P
Format Journal Article
LanguageEnglish
Published United States Elsevier Masson 15.01.2009
Subjects
Biochemistry, Molecular Biology
Calibration
Chromatography, High Pressure Liquid - methods
Cross-Linking Reagents - chemistry
Dipeptides - analysis
Humans
Life Sciences
Lysine - analysis
Mass Spectrometry - methods
Transglutaminases - chemistry
Transglutaminases - metabolism
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Summary:Transglutaminases catalyze the formation of Nepsilon-(gamma-glutamyl) isodipeptide crosslinks between proteins. These enzymes are thought to participate in a number of diseases, including neurological disease and cancer. A method associating liquid chromatography and multiple stage mass spectrometry has been developed for the simultaneous quantitation of [Nepsilon-(gamma-glutamyl) lysine] isodipeptide and lysine on an ion trap mass spectrometer. Highly specific detection has been achieved in MS3 mode. The method includes a derivatization step consisting of butylation of carboxylic groups and acetylation of amide groups, a liquid-liquid extraction, and a 19-min separation on a 100x2.1-mm Beta-basic C18 column with an acetonitrile gradient elution. 13C6-(15)N2 isotopes of the isodipeptide and the lysine serve as internal standards. The assay was linear in the range of 50 pmol/ml to 75 nmol/ml for the isodipeptide and the range of 10 nmol/ml to 3.5 micromol/ml for the lysine, with correlation coefficients greater than 0.99 for both ions. Intra- and inter-day coefficients of variation ranged from 3.5 to 15.9%. The method was successfully applied to human biological samples known to be crosslinked by transglutaminase such as cornified envelopes of epidermis, fibrin, and normal and Huntington disease brain.
Bibliography:ObjectType-Article-1
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ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2008.09.039