NOVEL PROTEOLYTIC PROTEIN AND PEPTIDE BIOMARKERS FOR TRAUMATIC BRAIN INJURY By GEORGE ANIS SARKIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 George Anis Sarkis To the souls and memory of my Father and Mother. To my Wife and Children thank you for your continued support, encouragement, and love throughout this process ACKNOWLEDGMENTS First and foremost, I would like to thank God, Jesus Christ and Mary Mother of God. You have given me the power to believe in my passion and pursue my dreams. I could never have done this without the faith I have in you, the Almighty. I thank my mother’s and father’s souls. I want to thank my family and friends who have stood by me as I have pursued my passion for learning. I thank my advisors, Professor Richard A. Yost and Professor. Kevin K.W. Wang, for giving me the opportunity to work with their research teams, and to all the team members. Besides my advisors, I would like to thank the rest of my committee, Professor Benjamin W. Smith, Professor Kenneth B. Wagener and Professor Kari B. Basso, for their insightful comments and encouragement, but also for the hard question, which incented me to widen my research from various perspectives. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 13 ABSTRACT ................................................................................................................... 17 CHAPTER 1 BACKGROUND AND SIGNIFICANCE ................................................................... 19 Introduction ............................................................................................................. 19 Military Based TBI ................................................................................................... 21 Sports-Based TBI ................................................................................................... 22 Current Methods of TBI Diagnosis .......................................................................... 23 Biomarker as Diagnosis Tools .......................................................................... 24 Biomarker Classifications ................................................................................. 26 The Detection of Biomarkers in Sports and Military-Related Injuries ................ 26 TBI Protein Biomarker............................................................................................. 27 Mass Spectrometry Based Peptidomics ................................................................. 30 2 EXPERIMENTAL METHODS AND ANALYTICAL DESIGNS ................................. 35 SDS-Gel Electrophoresis Combined with Immunoblotting ...................................... 35 Sample Preparation for Electrophoresis ........................................................... 35 Gel Electrophoresis .......................................................................................... 36 Coomassie Brilliant Blue Staining of Gels ........................................................ 37 Electrotransfer .................................................................................................. 37 Immunoblotting-Antibody Incubation and Detection ......................................... 38 Ultrafiltration Method ............................................................................................... 39 Mass Spectrometry ................................................................................................. 39 Reversed-Phase Liquid Chromatography Tandem Mass Spectrometry ........... 39 Velos Pro Dual-Pressure Linear Ion Trap Mass Spectrometer ......................... 41 3 IN VITRO DIGESTION OF PURIFIED HUMAN BRAIN PROTEINS AND NAÏVE MOUSE BRAIN LYSATE ........................................................................................ 45 5 The Biomarker Neurogranin .................................................................................... 45 The Biomarker Vimentin ......................................................................................... 47 Sample Preparation ................................................................................................ 47 Naïve Mouse Brain Lysate Preparation ............................................................ 48 In Vitro Digestion of Purified Protein-Naïve Mouse Brain Lysate ...................... 49 Analysis of Digested Samples ................................................................................ 49 Neurogranin ...................................................................................................... 49 Vimentin ........................................................................................................... 50 Naïve Mouse Brain Lysate ............................................................................... 50 Results and Discussion........................................................................................... 51 Purified Neurogranin Protein ............................................................................ 51 Purified Vimentin Protein .................................................................................. 52 Naïve Mouse Brain Lysate ...................................................................................... 53 Neurogranin ...................................................................................................... 53 Vimentin ........................................................................................................... 54 Conclusions ............................................................................................................ 54 4 NEURO-CELL CULTURE CYTOTOXIC CHALLENGES TO EXAMINE BRAIN PROTEOLYTIC PEPTIDES FORMATION ............................................................. 80 Introduction ............................................................................................................. 80 Cytotoxic Challenges .............................................................................................. 81 Cell Culture and Lysis ............................................................................................. 84 Results and Discussion........................................................................................... 85 The Primary Cortical Mixed Neuron-Astroglia (CTX)/ Neuro2a (N2a) cell line . 85 U-251 Cell Line, Human Glioblastoma Astrocytoma Cell ................................. 86 Conclusions ............................................................................................................ 87 5 MOUSE MODELS OF TRAUMATIC BRAIN INJURY ............................................. 99 Introduction ............................................................................................................. 99 In Vivo Models of Severe Traumatic Brain Injury .................................................. 102 In Vivo Models of Mild Traumatic Brain Injury ....................................................... 103 Mouse Brain Tissue Collection and Preparation ................................................... 103 Results and Discussion......................................................................................... 104 Immunoblotting Analysis for Cortex ................................................................ 104 Immunoblotting Analysis for Hippocampus..................................................... 104 Mass Spectrometry Analysis ................................................................................. 105 Conclusions .......................................................................................................... 105 6 6 ARCHIVED HUMAN TBI BIOFLUID CEREBROSPINAL FLUID .......................... 116 Human Bio-Samples Procurement ....................................................................... 116 Cerebrospinal Fluid Collection Protocol ......................................................... 117 Sample Preparation ........................................................................................ 118 Results and Discussion......................................................................................... 119 Conclusion ............................................................................................................ 120 7 SUMMARY AND FUTURE WORK ....................................................................... 128 Summary .............................................................................................................. 128 Future Work .......................................................................................................... 129 LIST OF REFERENCES ............................................................................................. 131 BIOGRAPHICAL SKETCH .......................................................................................... 142 7 LIST OF TABLES Table page 1-1 Glasgow coma scale showing different cases of TBI. ......................................... 33 1-2 Possible TBI markers (existing biomarkers)