An Effective Protocol for Proteome Analysis of Medaka (<i>Oryzias latipes</i>) 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...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2019-07-01
|
| Series: | Methods and Protocols |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2409-9279/2/3/66 |
| _version_ | 1818958419010256896 |
|---|---|
| author | Yeni Pérez-Gélvez Shem Unger Gerardo Gutiérrez-Sánchez Robert Bridger Olin E. Rhodes Carl Bergmann |
| author_facet | Yeni Pérez-Gélvez Shem Unger Gerardo Gutiérrez-Sánchez Robert Bridger Olin E. Rhodes Carl Bergmann |
| author_sort | Yeni Pérez-Gélvez |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-20T11:25:26Z |
| format | Article |
| id | doaj.art-18787500dd724cb495c85b15ce1bdc7b |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-12-20T11:25:26Z |
| publishDate | 2019-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Methods and Protocols |
| spelling | doaj.art-18787500dd724cb495c85b15ce1bdc7b2022-12-21T19:42:23ZengMDPI AGMethods and Protocols2409-92792019-07-01236610.3390/mps2030066mps2030066An Effective Protocol for Proteome Analysis of Medaka (<i>Oryzias latipes</i>) after Acute Exposure to Ionizing RadiationYeni Pérez-Gélvez0Shem Unger1Gerardo Gutiérrez-Sánchez2Robert Bridger3Olin E. Rhodes4Carl Bergmann5Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602, USADepartment of Biology, Wingate University, Wingate, NC 28174, USAExact Sciences Laboratories, 145 E Badger Rd Suite 100, Madison, WI 53713, USAComplex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602, USASavannah River Ecology Laboratory, The University of Georgia, Aiken, SC 29802, USAComplex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602, USAAll 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.https://www.mdpi.com/2409-9279/2/3/66in-gel digestionionizing radiationmedaka<i>Oryzias latipes</i>proteome |
| spellingShingle | Yeni Pérez-Gélvez Shem Unger Gerardo Gutiérrez-Sánchez Robert Bridger Olin E. Rhodes Carl Bergmann An Effective Protocol for Proteome Analysis of Medaka (<i>Oryzias latipes</i>) after Acute Exposure to Ionizing Radiation Methods and Protocols in-gel digestion ionizing radiation medaka <i>Oryzias latipes</i> proteome |
| title | An Effective Protocol for Proteome Analysis of Medaka (<i>Oryzias latipes</i>) after Acute Exposure to Ionizing Radiation |
| title_full | An Effective Protocol for Proteome Analysis of Medaka (<i>Oryzias latipes</i>) after Acute Exposure to Ionizing Radiation |
| title_fullStr | An Effective Protocol for Proteome Analysis of Medaka (<i>Oryzias latipes</i>) after Acute Exposure to Ionizing Radiation |
| title_full_unstemmed | An Effective Protocol for Proteome Analysis of Medaka (<i>Oryzias latipes</i>) after Acute Exposure to Ionizing Radiation |
| title_short | An Effective Protocol for Proteome Analysis of Medaka (<i>Oryzias latipes</i>) after Acute Exposure to Ionizing Radiation |
| title_sort | effective protocol for proteome analysis of medaka i oryzias latipes i after acute exposure to ionizing radiation |
| topic | in-gel digestion ionizing radiation medaka <i>Oryzias latipes</i> proteome |
| url | https://www.mdpi.com/2409-9279/2/3/66 |
| work_keys_str_mv | AT yeniperezgelvez aneffectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT shemunger aneffectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT gerardogutierrezsanchez aneffectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT robertbridger aneffectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT olinerhodes aneffectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT carlbergmann aneffectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT yeniperezgelvez effectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT shemunger effectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT gerardogutierrezsanchez effectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT robertbridger effectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT olinerhodes effectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation AT carlbergmann effectiveprotocolforproteomeanalysisofmedakaioryziaslatipesiafteracuteexposuretoionizingradiation |