University of New Orleans ScholarWorks@UNO University of New Orleans Theses and Dissertations Dissertations and Theses 5-20-2011 Patterns of protein expression in tissues of the killifish, undulusF heteroclitus and Fundulus grandis Naga Vijayalaxmi Abbaraju University of New Orleans Follow this and additional works at: https://scholarworks.uno.edu/td Recommended Citation Abbaraju, Naga Vijayalaxmi, "Patterns of protein expression in tissues of the killifish, undulusF heteroclitus and Fundulus grandis" (2011). University of New Orleans Theses and Dissertations. 113. https://scholarworks.uno.edu/td/113 This Dissertation-Restricted is protected by copyright and/or related rights. It has been brought to you by ScholarWorks@UNO with permission from the rights-holder(s). You are free to use this Dissertation-Restricted in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself. This Dissertation-Restricted has been accepted for inclusion in University of New Orleans Theses and Dissertations by an authorized administrator of ScholarWorks@UNO. For more information, please contact [email protected]. Patterns of protein expression in tissues of the killifish, Fundulus heteroclitus and Fundulus grandis A Dissertation Submitted to the Graduate Faculty of the University of New Orleans in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry by Naga Vijayalaxmi Abbaraju Bachelor of Science. Osmania University, India, 2001 Master of Science. Pune University, India, 2003 May, 2011 Dedication I would like to dedicate this dissertation to my beloved parents Jogeswara Prasad Abbaraju, Hemalatha Abbaraju, my husband Venkata Ramana Kethineedi, and my daughter Shriya Kethineedi. ii Acknowledgments It is a pleasure to thank many people who made this thesis possible. Foremost, I would like to express my sincere gratitude to my research advisor Dr. Bernard B. Rees for his support, guidance, and encouragement during graduate school. Working in Dr. Rees lab had been a wonderful learning experience; I could not have imagined having a better advisor and mentor for my PhD study. He has always been my inspiration for hardwork and perfection in whatever he does. I thank him for his patience, motivation, lots of research ideas, and good teaching throughout my tenure as a graduate student as well as in writing my thesis. I would like to thank Dr. Richard B. Cole who made by Chapter 4 possible. I thank him for all the knowledge I gained about mass spectrometry and database searching. I also thank Dr. Yang Cai for all the LC-MS/MS analysis and help with database search. I would like to express my gratitude to my other advisory committee members Dr. Zhengchang Liu, and Dr. Edwin Stevens for their comments and suggestions throughout my graduate research. I also would like to thank Dr. Guangdi Wang from Xavier University for use of the spotcutter and digester and Qiang Zhang for his help with the instruments. I greatly appreciate Kan Chen and Nazim Boutaghou for the training and help with the mass spectrometer and sample analysis. I thank my past and present research group members for their advice and help. I give special thanks to all the faculty and staff in the Departments of Chemistry and Biological Sciences for their support. I would like to greatly thank my family for their undying support and encouragement. I could not have made it this far without them. I thank my brother Sainath Abbaraju, my sister Srivalli Abbaraju, my father-in-law Narasimha Rao Kethineedi, and my mother-in-law, late Samanthakamani Kethineedi. iii Table of Contents List of Figures .................................................................................................................... vi List of Tables ................................................................................................................... viii Abstract ................................................................................................................................x Chapter 1 Introduction......................................................................................................1 1.1 Enzyme Activity versus Proteomics ..................................................................3 1.2 Protein Fractionation ..........................................................................................6 1.3 Mass Spectrometry.............................................................................................8 1.4 Protein Identification .......................................................................................10 1.5 Annotations ......................................................................................................16 1.6 Fish Proteomics ................................................................................................17 1.7 References ........................................................................................................19 Chapter 2 Effects of oxygen on glycolytic enzyme specific activities in liver and skeletal muscle of Fundulus heteroclitus ..................................................................................... 26 2.1 Abstract ............................................................................................................26 2.2 Introduction ......................................................................................................27 2.3 Materials and Methods .....................................................................................29 2.4 Results ..............................................................................................................35 2.5 Discussion ........................................................................................................38 2.6 Conclusions ......................................................................................................41 2.7 References. .......................................................................................................42 Chapter 3 Tissue sampling and protein recovery from tissues of the gulf killifish, Fundulus grandis ...............................................................................................................................45 3.1 Abstract ............................................................................................................45 3.2 Introduction ......................................................................................................46 3.3 Materials and Methods .....................................................................................47 3.4 Results ..............................................................................................................51 3.5 Discussion ........................................................................................................63 3.6 Conclusions ......................................................................................................65 3.7 References ........................................................................................................67 Chapter 4 2DE-MALDI-TOF/TOF tandem MS for evaluation of protein expression patterns in tissues of the model fish species, Fundulus grandis ...................................69 4.1 Abstract ............................................................................................................69 4.2 Introduction ......................................................................................................70 4.3 Materials and Methods .....................................................................................72 4.4 Results and Discussion ....................................................................................77 4.5 Conclusions ......................................................................................................97 4.6 References ........................................................................................................98 iv Chapter 5 Conclusions ...................................................................................................102 5.1 Future Directions ...........................................................................................103 5.2 References ......................................................................................................106 Appendix A .....................................................................................................................107 Appendix B .....................................................................................................................109 Appendix C .....................................................................................................................115 Appendix D .....................................................................................................................132 VITA................................................................................................................................141 v List of Figures Figure 1.1 Typical proteomics work flow ...........................................................................5 Figure 1.2 Representative peptide mass fingerprinting (PMF) and MS/MS spectra of the identified liver tissue proteins of Fundulus grandis (spot 8306) discussed in chapter 4. The matched peptides are