Application of a Label-free, Gel-free Quantitative Proteomics Method for Ecotoxicological Studies of Small Fish Species
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- 作者：K. J. Ralston-Hooper ; M. E. Turner ; E. J. Soderblom ; D. Villeneuve ; G. T. Ankley ; M. A. Moseley ; R. A. Hoke ; P. L. Ferguson
- 刊名：Environmental Science & Technology (ES&T)
- 出版年：2013
- 出版时间：January 15, 2013
- 年：2013
- 卷：47
- 期：2
- 页码：1091-1100
- 全文大小：415K
- 年卷期：v.47,no.2(January 15, 2013)
- ISSN：1520-5851
文摘
Although two-dimensional electrophoresis (2D-GE) remains the basis for many ecotoxicoproteomic analyses, newer 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, liquid chromatography鈥搕andem mass spectrometry (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 渭g of fadrozole/L of water for 4 days, and proteomic analysis was performed. Proteins were extracted and digested, and proteolytic peptides were separated via high-resolution one- or two-dimensional (1-D or 2-D) ultrapressure 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 by single-dimension UPLC. When multidimensional LC analysis (2-D) was performed, an average increase of 1.9脳 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.