An Effective Protocol for Proteome Analysis of Medaka (Oryzias latipes) after Acute Exposure to Ionizing Radiation

All terrestrial organisms are subject to evolutionary pressures associated with natural sources of ionizing radiation (IR). The legacy of human-induced IR associated with energy, weapons production, medicine, and research has changed the distribution and magnitude of these evolutionary pressures. To date, no study has systematically examined the effects of environmentally relevant doses of radiation exposure across an organismal proteome. This void in knowledge has been due, in part, to technological deficiencies that have hampered quantifiable environmentally relevant IR doses and sensitive detection of proteomic responses. Here, we describe a protocol that addresses both needs, combining quantifiable IR delivery with a reliable method to yield proteomic comparisons of control and irradiated Medaka fish. Exposures were conducted at the Savannah River Ecology Laboratory (SREL, in Aiken, SC), where fish were subsequently dissected into three tissue sets (carcasses, organs and intestines) and frozen until analysis. Tissue proteins were extracted, resolved by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and each sample lane was divided into ten equal portions. Following in-gel tryptic digestion, peptides released from each gel portion were identified and quantified by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain the most complete, comparative study to date of proteomic responses to environmentally relevant doses of IR. This method provides a simple approach for use in ongoing epidemiologic studies of chronic exposure to environmentally relevant levels of IR and should also serve well in physiological, developmental, and toxicological studies.

The Benefits of Long-Term Environmental Research, Friendships, and Boiled Peanuts

The Long-Term Ecological Research (LTER) program has enabled me to conduct more broadly relevant science by addressing questions within an interdisciplinary framework and to unravel the causes for surprising ecological phenomena through persistent studies and collaborations. Educational opportunities within the LTER program have connected me to students from grades K–12 to graduate levels in new ways from the field to the classroom, across places from Florida to Alaska, and among disciplines in a collaborative setting. The audience for my research expanded as a consequence of my experiences in the LTER program, and I have learned how to more effectively communicate integrative research to large audiences of scientists, policy-makers, and the public, often through nontraditional media. The LTER program is foremost a network of people, and I have found that science evolves most successfully when ideas and information are shared voluntarily across backgrounds, disciplines, and cultures in a network of cultivated, trusting relationships. The Florida Coastal Everglades (FCE) is the LTER site where I am currently the principal investigator, but the LTER program has been a part of my life for most of my career. My experiences in the LTER program began in the early 1990s when I was a graduate student at the University of Georgia, where the Coweeta (CWT) LTER site is based. Although I was not formally a part of CWT, many of my friends and professors were, so the program influenced my development as a scientist. I remember my first field trip to CWT, led by Gene Helfman and Judy Meyer, and the fun of snorkeling in mountain streams where we camped and conducted a few experiments, including examining the effects of rapid consumption of s’mores and boiled peanuts on preschool children (Judy and Gene’s kids). LTER-related activities wove in and out of my graduate student experience, and the rewards of sharing of ideas, data, friendships, and boiled peanuts created in me a lifelong commitment to persistent, collaborative science. This sense of fulfillment, of being part of something larger, was reinforced at the Savannah River Ecology Laboratory (SREL), where I conducted my research.