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Biomarker discovery and application in toxicological studies requires rapid and accurate methods to assess environmental risk. The emerging field of proteomics now includes powerful analytical tools involving mass spectrometry that can be utilized in toxicological studies. We are developing a focused proteomic technique using ESI- MS/MS to quantify the abundance of estrogen receptor (ER-alpha, ER-beta a, and ER-beta b) proteins in tissues of largemouth bass (LMB; Micropterus salmoides), a popular game fish that is used as a toxicological model. LMB ER sequences were cloned into expression vectors and recombinant proteins expressed for all three ER isoforms. Recombinant LMB ER proteins were purified to use as standards. In silico trypsin digestions using ProteinProspector and MS-Digest of each LMB ER isoform were compared to identify peptides unique to each LMB ER. The peptide 406-LIFAQDLILDR-418 distinguishes LMB ER-alpha from the other two LMB ER isoforms, and analysis with purified LMB ER-alpha digests shows that this peptide ionizes consistently. For quantification, this peptide was synthesized with heavy isotopes (2nd leucine) to produce a peptide with a mass of 1322.6 Da, a difference of approximately 6 Da when compared to the endogenous natural peptide mass of 1316.58 Da. A standard curve using the heavy isotope ER-alpha peptide showed linearity over five orders of magnitude and is detectable in the attomole range. Complex protein mixtures from the liver, gonad, and brain will be isolated using either whole-tissue protein or nuclear protein fractions for optimal compatibility with MS and ER peptide identification. In addition, Westerns blots are being developed for all three LMB ERs in parallel to determine the most sensitive method of ER detection. These techniques are powerful toxicological tools to rapidly identify changes in biomarker proteins such as ER isoforms.