PH04 Advances in Analysis and Measurements
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(PH011) Analysis of Vitellogenin in Fathead Minnow Plasma by MALDI-Mass Spectrometry.

Wunschel, D1, Schultz, I2, Skillman, A2, Wahl, K1, 1 Battelle Pacific Northwest National Laboratory, Analytical Chemistry, Richland, WA, USA2 Battelle Pacific Northwest National Laboratory , Marine Science Laboratory, Sequim, WA, USA

ABSTRACT- Vitellogenin (VTG) is widely used as a biomarker for exposure to estrogen and synthetic analogues in fish. It is also viewed as a critical endpoint in endocrine screening assays or tests. An important species in laboratory studies of endocrine disruptors is the fathead minnow (FHM), P. promelas . The principal methods for measuring VTG have been antibody-based assays such as ELISA. Despite its highly conserved nature across vertebrates, VTG antibodies can be poorly cross-reactive and typically require species specific antibodies to be raised for each fish species. Obtaining consistent quantitative results during inter-laboratory comparisons can also be challenging. Therefore, a complementary analytical method for measuring VTG would be desirable. Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been widely used for protein identification when coupled with an appropriate separation technique to reduce sample complexity. In this study we have coupled a simple cleanup step with an anion exchange HPLC — MALDI-MS method for measuring and identifying VTG in plasma samples. Several important aspects of studying VTG with MALDI-MS have been investigated prior to application of this method to FHM plasma samples. The method properly identified the HPLC fraction with VTG after an automated database search. Furthermore, the HPLC- UV measurement of VTG was able to detect differences in abundance similar to that found by an ELISA method.

Key words: fish, endocrine disruption, estrogen

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