Analysis of tissue proteomes of the Gulf killifish, Fundulus grandis, by 2D electrophoresis and MALDI-TOF/TOF mass spectrometry.

The Gulf killifish, Fundulus grandis, is a small teleost fish that inhabits marshes of the Gulf of Mexico and demonstrates high tolerance of environmental variation, making it an excellent subject for the study of physiological and molecular adaptations to environmental stress. In the present study, two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry were used to resolve and identify proteins from five tissues: skeletal muscle, liver, brain, heart, and gill. Of 864 protein features excised from 2D gels, 424 proteins were identified, corresponding to a 49% identification rate. For any given tissue, several protein features were identified as the same protein, resulting in a total of 254 nonredundant proteins. These nonredundant proteins were categorized into a total of 11 molecular functions, including catalytic activity, structural molecule, binding, and transport. In all tissues, catalytic activity and binding were the most highly represented molecular functions. Comparing across the tissues, proteome coverage was lowest in skeletal muscle, due to a combination of a low number of gel spots excised for analysis and a high redundancy of identifications among these spots. Nevertheless, the identification of a substantial number of proteins with high statistical confidence from other tissues suggests that F. grandis may serve as a model fish for future studies of environmental proteomics and ultimately help to elucidate proteomic responses of fish and other vertebrates to environmental stress.

Effects of dissolved oxygen on glycolytic enzyme specific activities in liver and skeletal muscle of Fundulus heteroclitus.

Many aquatic habitats are characterized by variable concentrations of dissolved oxygen (DO), and fish that occur in these habitats respond to changes in DO through behavioral, physiological, and biochemical adjustments. The goal of the present study was to measure the effects of an ecologically relevant range of DO treatments, from severe hypoxia to moderate hyperoxia, on the maximal activities of nine glycolytic enzymes during chronic exposure of the mummichog, Fundulus heteroclitus. Over the 28 days of exposure period, specific activity was significantly affected by DO for three enzymes in liver and one enzyme in white skeletal muscle, although at specific times of exposure three other muscle enzymes were affected by DO. In general, exposure of fish to severe hypoxia led to higher specific activities in liver, but lower specific activities in skeletal muscle. Exposure to hyperoxia did not elicit changes in enzyme specific activities in either tissue. Surprisingly, exposure duration had strong effects on glycolytic enzyme specific activities in both liver and white skeletal muscle, with specific activities increasing with exposure duration regardless of DO treatment. The results demonstrate that the effects of DO on enzyme specific activities were restricted to a subset of the glycolytic enzymes in liver and white skeletal muscle of F. heteroclitus and that the directions of the changes were opposite in these two tissues. These observations suggest that the mechanisms resulting in these alterations are enzyme- and tissue specific, rather than applying uniformly to all enzymes within the glycolytic pathway.

Protein recovery and identification from the gulf killifish, Fundulus grandis: comparing snap-frozen and RNAlater® preserved tissues.

Reliable proteomic analysis of biological tissues requires sampling approaches that preserve proteins as close to their in vivo state as possible. In the current study, the patterns of protein abundance in one-dimensional (1-D) gels were assessed for five tissues of the gulf killifish, Fundulus grandis, following snap-freezing tissues in liquid nitrogen or immersion of fresh tissues in RNAlater(®). In liver and heart, the protein profiles in 1-D gels were better preserved by snap-freezing, while in gill, the 1-D protein profile was better preserved by immersion in RNAlater(®). In skeletal muscle and brain, the two approaches yielded similar patterns of protein abundance. LC-MS/MS analyses and database searching resulted in the identification of 17 proteins in liver and 12 proteins in gill. Identified proteins include enzymes of energy metabolism, structural proteins, and proteins serving other biological functions. These protein identifications for a species without a sequenced genome demonstrate the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.