Method for detection and quantitation of fathead minnow vitellogenin (Vtg) by liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Abstract

Vitellogenin (Vtg) is a well-recognized biomarker of estrogen exposure in many species, particularly fish. This large protein shares a high degree of sequence homology across a large number of species. Quantitative measurement is currently done using antibody-based assays. These assays frequently require purification of Vtg and antibody production from each species because there is poor cross reactivity between antibodies for different fish. Therefore, complementary methods of measuring Vtg are desirable. Mass spectrometric (MS) analysis coupled to database searching offers the promise of a general method for protein identification. In this study, we used the well-characterized Vtg from rainbow trout ( Oncorhynchus mykiss) to evaluate the analytical parameters for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the intact protein. The low sensitivity observed for the intact protein demonstrated that a proteolytic digestion would be necessary for MALDI-MS identification of Vtg. An analytical scale high performance liquid chromatography (HPLC) separation combined with MALDI-MS was used to measure and confirm the identity of Vtg from the plasma of an important species for regulatory agencies, fathead minnow ( Pimephales promelas). The small volume requirement of this method (<10 μL) was found to be compatible with the plasma volume obtained from a few minnows. Importantly, the HPLC–MALDI-MS measurement of fathead minnow Vtg abundance after exposure to estradiol was similar to previously obtained enzyme linked immunosorbant assays (ELISA) data.