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Identification of two Isoforms of Vitelline Envelope Protein as Complementary Biomarkers to Vitellogenin in the Plasma of Rainbow Trout Exposed to 17beta-estradiol
Citation:
SERRANO, J. A., L. HIGGINS, C. C. WALKER, M. J. HEMMER, AND K. SELINAS. Identification of two Isoforms of Vitelline Envelope Protein as Complementary Biomarkers to Vitellogenin in the Plasma of Rainbow Trout Exposed to 17beta-estradiol. Presented at SETAC Annual Meeting, Tampa, FL, November 19, 2008.
Impact/Purpose:
The two vitelline isoforms identified herein validate the protein masses found during the high throughput, low resolution screening process as estrogen-responsive biomarkers and serve as effective complements to vitellogenin as indicators to estradiol exposure in aquatic species.
Description:
In the present study, protein markers of estrogenic exposure in rainbow trout (Oncorhynchus mykiss) were isolated and identified using innovative sample preparation techniques followed by advanced MS and bioinformatics approaches. Juvenile trout were administered 17ß-estradiol through dietary exposure. Plasma from exposed and control trout was analyzed by high throughput, low resolution MALDI TOF MS. Ten fragments (m/z 3248.3 3834.5) were found differentially regulated in exposed specimens. To isolate proteins of interest from plasma matrix, samples were fractionated using solid-phase ligand libraries, fragmented and spectral data acquired using MALDI tandem MS. A protein fragment at m/z 3307.7 was identified as a significant (p<0.05) match for vitelline envelope protein alpha using MASCOT®. An additional estrogen-responsive protein was further purified via strong anion exchange spin column fractionation, cassette dialysis concentration, detergent removal and HPLC separation. Fragmentation spectra were obtained by both Tempo™LC -MALDI TOF-TOF and electrospray tandem MS. Protein identification was performed by automated and manual de Novo sequencing and MS BLAST search. A fragment at m/z 3248.3 was identified with high confidence as a match for vitelline envelope protein gamma. Overall, the two vitelline isoforms identified herein validate the protein masses found during the high throughput, low resolution screening process as estrogen-responsive biomarkers and serve as effective complements to vitellogenin as indicators to estradiol exposure in aquatic species.