Quantitative measurement of fathead minnow vitellogenin by liquid chromatography combined with tandem mass spectrometry using a signature peptide of vitellogenin.

Abstract

Vitellogenin (VTG) has been proposed as a sensitive biomarker of exposure to environmental estrogenic contaminants that induce VTG production in oviparous species. Enzyme-linked immunosorbent assay (ELISA) methods are currently widely used to measure the VTG levels. In this paper, a new liquid chromatography combined with tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative analysis of VTG in the plasma of fathead minnows exposed to 17alpha-ethinylestradiol (EE2) has been developed. This method includes, first, the selection of the signature peptide, which involves sodium dodecyl sulfate-polyarylamide gel electrophoresis separation, in-gel digestion, LC/ESI-MS/MS analysis with an ion trap mass spectrometer, and sequence determination with the TurboSEQUEST MS/MS database application; second, optimization of the selected signature peptide in multireaction monitor (MRM) mode with a triple quadrupole mass spectrometer; and third, trypsin digestion of plasma and VTG quantitation via MRM-mode LC/ESI-MS/MS. A series of plasma samples from fathead minnows following exposure to EE2 was assayed. A good correlation was found when EE2-induced plasma samples from fathead minnows were analyzed with ELISA and the described new method. Although used here with fathead minnow, the new LC/ESI-MS/MS method could be easily applied to the analysis of VTG expressed in any other fish species. Quantitation of VTG by this method was found to be highly specific and linear. The absence of potential artifactual measurements of VTG at low exposure levels could also be critical in future studies that evaluate weakly estrogenic compounds in aquatic species.