Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

J.S.B. de Vlieger, A.J. Kolkman, K.A.M. Ampt, J.N.M. Commandeur, N.P.E. Vermeulen, J. Kool, S.S. Wijmenga, W.M.A. Niessen, H. Irth, M. Honing

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)α and hERβ integrating target-ligand interactions and liquid chromatography-high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D-
Original languageEnglish
Pages (from-to)667-674
Number of pages8
JournalJournal of Chromatography B
Volume878
Issue number7-8
DOIs
Publication statusPublished - 2010

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