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Identification of candidate biomarker proteins by sequence identity

One of the aims of the EASYRING project was to identify new biomarker candidates to endocrine disrupting compounds in fish and amphibians. Carp (Cyprinus carpio) and African clawed frog (Xenopus laevis) were selected as test organism for in in vivo exposures by four model compounds, ethynylestradiol (EE2), tamoxifen (TAM), metyldihydrotestosterone (MDHT) and flutamide (FLU).

Protein patterns in of different tissues (liver, plasma and mucus) were analysed by 2DE and compared with control samples. Proteins with changed levels and/ or altered pI were excised from the gels, trypsinised and analyzed by MALDI-TOF or LC-MS/MS for identification by database searches using mass-lists or unassigned MS spectra. MALDI-TOF MS-spectra were calibrated and processed with FlexAnalysis and Biotools (Bruker Daltonics) for database-searches in NCBInr using Mascot (Matrix Science). For analyses with LC-MS/MS data merge (mgf) files of the MS/MS spectra were used for peptide searches in NCBInr using Mascot and Phenyx (GeneBio). Hypotetical proteins of unknown identity and function were subjected to Blastp searches for identification.

After 2DE analyses of carp plasma samples, we chose 3 proteins as candidate biomarker proteins for endocrine disruption in carp exposed to estrogenic compounds (64 ng/l EE2). These were: 1-antitrypsin (A1AT), transferrin (TRA) and warm-temperature-acclimation-related-65 kDa-protein (WTA-65). We have selected two antigens to each protein based on different criteria for peptide selection for antibody production. Six carp specific polyclonal antibodies have been produced ands these are being tested and validated as possible new biomarker candidates.

Xenopus laevis were exposed to EE2, TAM, MDHT, FLU (all 10-8 M) and 2-DE analysis was performed on pooled samples from each treatment group. Males and females were analyzed separately and compared to controls. Estrogen regulated protein Ep45 was found to be a new candidate biomarker protein for estrogenic exposure to Xenopus. The proteins specifically repressed by MDHT treatment were identified as enolase, creatine kinase, endodermin and pentraxin fusion protein precursor. In response to FLU treatment two isoforms of albumin (68 and 74 kDa) appeared to be specifically upregulated in both male and female samples.
Of the identified proteins in Xenopus liver after exposure to EE2, oxidative stress related proteins were upregulated. These include Hsp70, Hsp90, catalase and thioredoxin.

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University of Bergen
Dept of Molecular Biology, PO Box 7800
N-5020 Bergen
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