Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Garrison L. Birch Entitled LIPIDOMIC PROFILING OF DICTYOSTELIUM DISCOIDEUM Master of Science For the degree of Is approved by the final examining committee: Robert E. Minto Chair Brenda J. Blacklock Michael J. McLeish To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________Robert E. Minto ____________________________________ Approved by: Martin J. O'Donnell 11/14/2011 Head of the Graduate Program Date Graduate School Form 20 (Revised 9/10) PURDUE UNIVERSITY GRADUATE SCHOOL Research Integrity and Copyright Disclaimer Title of Thesis/Dissertation: LIPIDOMIC PROFILING OF DICTYOSTELIUM DISCOIDEUM For the degree of MasterChoose ofyour Science degree I certify that in the preparation of this thesis, I have observed the provisions of Purdue University Executive Memorandum No. C-22, September 6, 1991, Policy on Integrity in Research.* Further, I certify that this work is free of plagiarism and all materials appearing in this thesis/dissertation have been properly quoted and attributed. I certify that all copyrighted material incorporated into this thesis/dissertation is in compliance with the United States’ copyright law and that I have received written permission from the copyright owners for my use of their work, which is beyond the scope of the law. I agree to indemnify and save harmless Purdue University from any and all claims that may be asserted or that may arise from any copyright violation. Garrison L. Birch ______________________________________ Printed Name and Signature of Candidate 11/14/2011 ______________________________________ Date (month/day/year) *Located at http://www.purdue.edu/policies/pages/teach_res_outreach/c_22.html LIPIDOMIC PROFILING OF DICTYOSTELIUM DISCOIDEUM A Thesis Submitted to the Faculty of Purdue University by Garrison L. Birch In Partial Fulfillment of the Requirements for the Degree of Master of Science December 2011 Purdue University Indianapolis, Indiana ii ACKNOWLEDGMENTS I am very grateful to the many people that encouraged and helped me along the journey of pursuing this degree. I would especially like to thank my advisor Dr. Brenda Blacklock for her wisdom and guidance. I also had the great pleasure of working with Dr. Robert Minto, who allowed me to collaborate on his research and gave me sound advice and support throughout my graduate career. I would also like to thank my committee members for their willingness to see me through this challenging process. Dr. Karl Dria and Mr. Cary Pritchard have been invaluable assets throughout my career. I would like to thank my lab co-workers, and particularly Michael Shepard, for their help, advice, and friendship. iii TABLE OF CONTENTS Page LIST OF TABLES ................................................................................................. v LIST OF FIGURES ...............................................................................................vi LIST OF ABBREVIATIONS .................................................................................. x ABSTRACT ........................................................................................................ xiii CHAPTER 1. INTRODUCTION ............................................................................ 1 1.1. Fatty acid elongation and Dictyostelium discoideum .................................. 1 1.2. Fatty acid biosynthesis and degradation .................................................... 6 1.2.1. Fatty Acid Biosynthesis ....................................................................... 7 1.2.2. Fatty Acid Degradation ...................................................................... 11 1.3. Sphingolipids ............................................................................................ 14 1.3.1. Sphingolipid Biosynthesis .................................................................. 17 CHAPTER 2. FATTY ACID PROFILING OF DICTYOSTELIUM DISCOIDEUM ..................................................................................................... 22 2.1. Introduction .............................................................................................. 22 2.2. Methods and materials ............................................................................. 23 2.2.1. Maintenance and growth of Dictyostelium discoideum ...................... 24 2.2.2. Preparation and Analysis of Derivatives ............................................ 24 2.3. Results and discussion ............................................................................ 26 2.3.1. Synthesis of authentic fatty acid standards ....................................... 26 2.3.2. The fatty acid profile of D. discoideum ............................................... 31 CHAPTER 3. FATTY ACID METABOLISM IN DICTYOSTELIUM DISCOIDEUM ..................................................................................................... 41 3.1. Introduction .............................................................................................. 41 3.2. Methods and materials ............................................................................. 43 3.2.1. Calculation of incorporation levels ..................................................... 43 3.2.2. Statistical analysis ............................................................................. 45 3.3. Results and discussion ............................................................................ 46 3.3.1. Synthesis of deuterium-labeled fatty acids ........................................ 46 3.3.2. In vivo feeding studies ....................................................................... 52 iv Page CHAPTER 4. SPHINGOLIPID CHARACTERIZATION IN DICTYOSTELIUM DISCOIDEUM ..................................................................................................... 74 4.1. Introduction .............................................................................................. 74 4.2. Methods and materials ............................................................................. 74 4.2.1. Culturing of D. discoideum and S. cerevisiae .................................... 75 4.2.2. Ba(OH)2 hydrolysis ............................................................................ 75 4.2.3. Dinitrophenyl derivatization of LCBs .................................................. 76 4.2.4. Total lipid extraction ........................................................................... 76 4.2.5. Mild alkaline hydrolysis ...................................................................... 77 4.2.6. Lithiation of sphingolipids .................................................................. 77 4.2.7. LC-MS analysis of lipids .................................................................... 78 4.3. Results and discussion ............................................................................ 82 4.3.1. Identification of sphingolipid long chain bases in D. discoideum ....... 82 4.3.2. Isolation of sphingolipids for characterization .................................... 93 4.3.3. Sphingolipid characterization for D. discoideum ................................ 98 4.3.4. Closing Remarks ............................................................................. 113 CHAPTER 5. EXPERIMENTAL DETAILS OF THE SYNTHESIS OF FATTY ACIDS .................................................................................................. 115 5.1. General experimental information .......................................................... 115 5.2. Synthetic standards for characterization of fatty acid profile .................. 116 5.2.1. Synthesis of 18:2 5,9 (35) ................................................................. 116 5.3. Production of 2H-labeled fatty acids for in vivo studies........................... 122 5.3.1. Synthesis of stearic acid-17,17,18,18,18-d5 (49) ............................. 123 5.3.2. Synthesis of stearic acid-3,3,4,4-d4 (55) .......................................... 127 5.3.3. Synthesis of octadec-5-enoic acid-16,16,17,17,18,18,18-d7 (74) .... 131 5.3.4. Synthesis of octadec-11-enoic acid-16,16,17,17,18,18,18-d7 (81) .. 137 5.4. Synthesis of 2H-labeled fatty acid stereoisomers ................................... 140 REFERENCES ................................................................................................. 152 APPENDIX ....................................................................................................... 163 v LIST OF TABLES Table Page Table 1. Summary of alkylations and the resulting products. ............................. 29 Table 2. GC-MS retention time comparison of methyl ester and pyrrolidine- derivatized synthetic standards to the FAs found in D. discoideum ...... 32 Table 3. Optimizer defined conditions for MRM. ................................................. 80 Table 4. Three long chain bases quantified by MRM as free LCBs .................... 88 Table 5. The sphingolipids of D. discoideum .................................................... 114 vi LIST OF FIGURES Figure Page Figure 1. Relevant lipid structures; a monounsaturated C18 fatty acid, a phospholipid, and the sphingolipids