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Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species
Citation:
Ralston-Hooper, K., M. Turner, E. Soderblom, D. Villeneuve, G. Ankley, M. Moseley, R. Hoke, AND P. Ferguson. Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species. ENVIRONMENTAL SCIENCE AND TECHNOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, 47(2):1091-1100, (2013).
Impact/Purpose:
Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, new, non gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehensive, LC-/MS/MS-based proteomic approach to identify and quantify differentially expressed hepatic proteins from female fathead minnows exposed to fadrozole, a potent inhibitor of estrogen synthesis. Female fathead minnows were exposed to 0 (control), 0.04, and 1.0 µg fadrozole/L water for 4 d and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides separated via high resolution 1-dimensional or 2-dimensional ultra-pressure liquid chromatography (UPLC) and analyzed by tandem mass spectrometry. Mass spectra were searched against the National Center for Biotechnology Information (NCBI) ray-finned fish (Actinopterygii) database, resulting in identification of 782 unique proteins using single-dimension UPLC. When multi-dimensional LC analysis (2-D) was performed, an average increase of 1.9X in the number of identified proteins was observed. Differentially expressed proteins in fadrozole exposures were consistent with changes in liver function, including a decline in concentrations of vitellogenin as well as other proteins associated with endocrine function and cholesterol synthesis. Overall, these results demonstrate that a gel-free, label-free proteomic analysis method can successfully be utilized to determine differentially expressed proteins in small fish species after toxicant exposure.
Description:
Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, new, non gel-based methods are beginning to be applied to overcome throughput and coverage limitations of 2D-GE. The overall objective of our research was to apply a comprehensive, LC-/MS/MS-based proteomic approach to identify and quantify differentially expressed hepatic proteins from female fathead minnows exposed to fadrozole, a potent inhibitor of estrogen synthesis. Female fathead minnows were exposed to 0 (control), 0.04, and 1.0 µg fadrozole/L water for 4 d and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides separated via high resolution 1-dimensional or 2-dimensional ultra-pressure liquid chromatography (UPLC) and analyzed by tandem mass spectrometry. Mass spectra were searched against the National Center for Biotechnology Information (NCBI) ray-finned fish (Actinopterygii) database, resulting in identification of 782 unique proteins using single-dimension UPLC. When multi-dimensional LC analysis (2-D) was performed, an average increase of 1.9X in the number of identified proteins was observed. Differentially expressed proteins in fadrozole exposures were consistent with changes in liver function, including a decline in concentrations of vitellogenin as well as other proteins associated with endocrine function and cholesterol synthesis. Overall, these results demonstrate that a gel-free, label-free proteomic analysis method can successfully be utilized to determine differentially expressed proteins in small fish species after toxicant exposure.