Mass Spectrometric Analysis of Neurologically-Relevant Molecules Item Type text; Electronic Dissertation Authors Smith, Catherine L. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 03/10/2021 20:43:26 Link to Item http://hdl.handle.net/10150/626689 MASS SPECTROMETRIC ANALYSIS OF NEUROLOGICALLY-RELEVANT MOLECULES by Catherine L. Smith ______________________________________________ Copyright © Catherine L. Smith 2018 A Dissertation Submitted to the Faculty of the DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN CHEMISTRY In the Graduate College THE UNIVERSITY OF ARIZONA 2018 2 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: Catherine L. Smith 3 DEDICATION For Brandon I couldn’t have done this without you. 4 TABLE OF CONTENTS TITLE PAGE LIST OF FIGURES 13 LIST OF TABLES 17 LIST OF ABBREVIATIONS 18 LIST OF CONTRIBUTORS 21 ABSTRACT 22 CHAPTER 1. Introduction: Quantifying neurochemicals with mass 23 spectrometry 1.1 Introduction 23 1.2 Sampling and analysis of neurochemicals 25 1.2.1 Ex situ 26 1.2.1.1 Affinity assays 26 1.2.1.2 Separation and detection techniques 27 1.2.2 In vitro 30 1.2.2.1 Biological fluids 30 1.2.2.2 Cell models 31 1.2.3 In vivo 31 1.2.3.1 Direct measurement techniques 32 1.2.3.2 Microdialysis 32 1.3 Tandem mass spectrometry 36 1.4 Neurotransmission in insects 42 1.4.1 Classical neurotransmission 43 1.4.2 Quantitation of neurotransmitters 46 5 1.5 Drug penetration of the blood-brain barrier 47 1.5.1 Mechanisms of BBB penetration 48 1.5.2 Peptide-based drug development 50 1.5.3 Current measurement techniques and challenges 51 1.6 Overview 53 CHAPTER 2. Separation-free quantitation of biogenic amines in 55 brain tissue with mass spectrometry 2.1 Introduction 56 2.2 Materials and methods 59 2.2.1 Chemicals and reagents 59 2.2.2 Rats 59 2.2.3 Unilateral 6-hydroxydopamine (6-OHDA) lesion 59 method 2.2.4 Amphetamine-induced rotation 60 2.2.5 Rat tissue collection and preparation 60 2.2.6 Drosophila melanogaster 60 2.2.7 Apis mellifera 61 2.2.8 Derivatization reaction 62 2.2.9 Mass spectrometric detection and quantitation 63 2.3 Results and Discussion 63 2.3.1 Mass spectrometric detection of labeled biogenic 63 amines 2.3.2 Comparison to “gold standard” LC-EC 75 2.3.3 Quantification of biogenic amines in insect brain 80 homogenate 6 2.3.4 Sexual dimorphism was not detected across three 85 Drosophila strains 2.3.5 Deletion of ABC transporter does not affect 87 whole-head histamine 2.3.6 Genetic inactivation of DAT in Drosophila 89 melanogaster does not systematically alter whole- head biogenic amine content 2.4 Conclusions 90 2.5 Author contributions 90 2.6 Acknowledgments 91 CHAPTER 3. Nosema ceranae parasitism in honey bees (Apis 92 mellifera) increases biogenic amines associated with foraging behavior and alters olfactory learning and memory 3.1 Introduction 93 3.2 Materials and Methods 95 3.2.1 Animals 95 3.2.2 Feeding 95 3.2.3 Nosema inoculum 96 3.2.4 Spore counts 97 3.2.5 Learning and memory 97 3.2.6 Amino acid analysis of brain tissue and pollen 98 3.2.7 Biogenic amine analysis of brain tissue 101 3.2.8 Statistics 102 3.3 Results and Discussion 102 7 3.3.1 Odor-associative learning and memory in nurse- 102 and forager-aged bees 3.3.2 Amino acid concentrations in the whole brain of 106 nurse- and forager-aged bees 3.3.3 Biogenic amine levels in the whole brain of nurse- 112 and forager-aged bees 3.3.4 Comparing whole-brain content of compounds 118 along biological synthesis pathways can elucidate mechanisms of change in infected bees 3.4 Conclusions 122 3.5 Author Contributions 123 3.6 Acknowledgments 124 CHAPTER 4. Glycosylation of peptide-based drug candidates 125 improves in vivo stability and penetration of the blood-brain barrier 4.1 Introduction 126 4.2 Materials and Methods 131 4.2.1 Chemicals and reagents 131 4.2.2 Peptide synthesis 131 4.2.3 MS identification and quantification of peptides 131 4.2.4 In vitro stability of peptides 133 4.2.5 Animals 133 4.2.6 Carotid artery catheterization 133 4.2.7 Microdialysis surgery 134 4.2.8 Microdialysis and blood draws 135 8 4.3 Results and Discussion 135 4.3.1 High ion throughput of glycosylated peptides 135 during tandem MS yields lower limits of detection 4.3.2 Glycosylated compounds have improved in vitro 144 stability 4.3.3 “Shotgun microdialysis” for direct comparison of 149 in vivo lifetime and BBB penetration of peptide derivatives 4.4 Conclusions 152 4.5 Author Contributions 154 4.6 Acknowledgements 155 CHAPTER 5. Identification of optimal structural modifications for 156 the improvement of peptide-based drug delivery properties: an Angiotensin 1-7 study of in vivo stability and BBB penetration 5.1 Introduction 157 5.2 Materials and Methods 161 5.2.1 Chemicals and reagents 161 5.2.2 Animals 161 5.2.3 Peptide synthesis 162 5.2.4 Mass spectrometric identification and 162 quantification of compounds 5.2.5 In vitro stability of Angiotensin derivatives. 162 5.2.6 Carotid artery catheterization 164 5.2.7 Microdialysis surgery 165 9 5.2.8 Microdialysis and blood draws 165 5.3 Results and Discussion 166 5.3.1 Mass spectrometric identification and 166 quantification of compounds 5.3.2 Modified compounds have increased in vitro 175 lifetime 5.3.3 Non-selective protein binding may decrease free 179 fraction of peptides 5.3.4 “Shotgun microdialysis” allows for the direct 181 comparison of in vivo lifetime and BBB penetration of Ang 1-7 derivatives 5.4 Conclusions 192 5.5 Author Contributions 193 5.6 Acknowledgments 193 CHAPTER 6. Conclusions and future directions 194 6.1 Expanding the portfolio of BzCl-DI-MS 195 6.1.1 Increasing the number of amine compounds 195 being investigated 6.1.2 Application to additional factors in honey bee hive 196 health 6.2 Glycosylation and other modifications for improved 200 pharmaceutical properties of peptide-based drugs 6.2.1 Investigation of apparent PACAP degradation 202 10 6.2.2 Positive and negative controls for blood-brain 205 barrier penetration will strengthen our methodology 6.3 Potential routes for the elucidation of enkephalin 207 dynamics in vivo 6.3.1 Quantitation of enkephalins in chronic pain 209 models 6.3.2 Pharmaceutical probing of enkephalin dynamics 209 6.4 Concluding remarks 211 APPENDIX A Procedure: Benzoyl Chloride Labeling Reaction 214 APPENDIX B Quantification of endogenous opioid peptide 216 dynamics in the anterior cingulate cortex by online- preservation microdialysis B.1 Introduction 217 B.2 Materials and Methods 219 B.2.1 Chemicals 219 B.2.2 Statistical analysis & validation criteria 219 B.2.3 Animals 220 B.2.4 Intracranial (ACC) cannula implantation for 221 microdialysis B.2.5 CSF collection from cisterna magna for in vitro 221 experiments B.2.6 In vivo microdialysis procedure 222 B.2.7 Online-preservation system 223 3 B.2.8 Sample preparation for nano LC-MS analysis 224 11 B.2.9 Nano LC-MS3 analysis of opioids 224 B.2.10 Flow-injection MS study of opioid degradation 225 B.3 Results and Discussion 226 B.3.1 Chip-based ESI for improved nano LC-MS3 226 quantification of endogenous opioids B.3.2 Initial in vivo measurements in the ACC and 231 degradation of opioid peptides B.3.3 Online-preservation of endogenous peptides 237 B.3.4 Online-preservation microdialysis allowed 239 quantifiable measurement of EOPs in the ACC B.3.5 Identification and quantification of methionine 239 enkephalin sulphoxide B.3.6 Endomorphin II is present in dialysate from the 241 ACC B.3.7 Stimulated release of enkephalins in the ACC 241 B.4 Conclusions 244 B.5 Author Contributions 246 B.6 Acknowledgements 246 APPENDIX C Compound structures, mass spectra, and 247 chromatograms REFERENCES 261 12 LIST OF FIGURES FIGURE TITLE PAGE 1.1 Principles of microdialysis 34 1.2 Principles of electrospray ionization 38 1.3 Neural transmission 45 1.4 Mechanisms of penetration of the blood-brain barrier 49 2.1 Schotten-Baumann reaction mechanism 64 2.2 Benzoyl chloride derivitization 65 2.3 Workflow for sample preparation 67 2.4 MS2 spectra for target compounds 68 2.5 Representative calibration curves 69 2.6 Labeled compounds show stability against oxidation 74 2.7 Comparison to existing LC-EC method for validation of 77 quantitation 2.8 Dopamine and serotonin correlations between methods 79 2.9 Biogenic amine quantification in honey bees matches previously 81 reported results 2.10 Biogenic amine content (ng/head) in Drosophila melanogaster 86 whole heads, by sex 2.11 Biogenic amine content (ng/head) in Drosophila melanogaster 88 whole heads, by strain 3.1 Honey bee learning and memory trials 104 3.2 N.