title: Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species.
abstract: 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-tandem 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 μg 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× 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.
authors.affiliation: Nicholas School of the Environment, Duke University, Durham, North Carolina 27708, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Ralston-Hooper
authors.forename: M E
authors.initials: ME
authors.lastname: Turner
authors.forename: E J
authors.initials: EJ
authors.lastname: Soderblom
authors.forename: D
authors.initials: D
authors.lastname: Villeneuve
authors.forename: G T
authors.initials: GT
authors.lastname: Ankley
authors.forename: M A
authors.initials: MA
authors.lastname: Moseley
authors.forename: R A
authors.initials: RA
authors.lastname: Hoke
authors.forename: P L
authors.initials: PL
authors.lastname: Ferguson
mesh_terms: D000818:Animals; D047072:Aromatase Inhibitors / Q000633:toxicity*; D002853:Chromatography, Liquid / Q000379:methods; D003530:Cyprinidae / Q000378:metabolism*; D054750:Ecotoxicology / Q000379:methods; D017316:Fadrozole / Q000633:toxicity*; D005260:Female; D029941:Fish Proteins / Q000302:isolation & purification / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D047072:Aromatase Inhibitors; D029941:Fish Proteins; D020543:Proteome; D014874:Water Pollutants, Chemical; D017316:Fadrozole