ANNOTATION OF THE HUMAN ODONTOBLAST CELL LAYER AND DENTAL PULP PROTEOMES AND N-TERMINOMES by Simon Abbey B.Sc., Queen’s University, 1989 D.M.D., The University of British Columbia, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Craniofacial Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2017 © Simon Abbey, 2017 Abstract The proteome and N-terminome of the human odontoblast cell layer was identified for the first time by shotgun proteomic and terminal amine isotopic labeling of substrates (TAILS) N-terminomic analyses, respectively, and compared with that of human dental pulp stroma from 3rd molar teeth. After reverse-phase liquid chromatography-tandem mass spectrometry, >170,000 spectra from the shotgun and TAILS analyses were matched by four search engines to 4,888 and 12,063 peptides in the odontoblast cell layer and pulp stroma, respectively. Using the Trans-Proteomic Pipeline, I identified 895 and 2,423 unique proteins in these tissues at an FDR of ≤ 1 %. In the odontoblast cell layer proteome I found proteomic evidence for dentin sialophosphoprotein, which is cleaved into dentin phosphoprotein and dentin sialoprotein, proteins that are important in dentin mineralization. Further, 222 proteins of the odontoblast cell layer were not found in the pulp, suggesting many of these proteins are synthesized preferentially by odontoblasts. I also found minor differences in the odontoblast cell layer between the dental pulp proteomes of older and younger donors. The human dental pulp stroma proteome was expanded by 974 new proteins, not previously identified among the 4,123 proteins identified in our previous dental pulp study (Eckhard et al., 2015). Thus, by exploring the proteome of the odontoblast cell layer and expanding the known dental pulp proteome, we found distinct proteome differences when compared with each other and with dentin. The mass spectrometry raw data and metadata have been deposited to ProteomeXchange with the PXD identifier ˂PXD006557˃. ii Lay Summary Dental pulp comprises the central pulpal stroma and the odontoblast periphery. We hypothesized each expresses different proteins. We removed dental pulp tissue from freshly extracted teeth and separated these layers. Protein from each of these samples was broken down into its constituent peptides and these were identified using mass spectrometry. Using software parent proteins were identified from peptides which were unique to individual human proteins. We thus identified 4,888 peptides in the odontoblast sample and 12,063 peptides in the pulp stroma. 222 proteins were found only in the odontoblast sample, suggesting they are synthesized by odontoblasts. We also found differences in the odontoblast cell layer proteomes between older and younger donors. In summary we expanded the human dental pulp proteome by 974 new proteins not previously found in our previous dental pulp study. We also found proteome differences between the odontoblast cell layer and the pulp stroma. iii Preface Dr. Overall and I met in early 2014 to discuss this research project. The Overall lab is involved in the human proteome project and Dr. Overall was interested in taking me on as a graduate student to do proteomics on human dental pulp, adding this tissue to others already being studied in the lab. The terminal amine isotopic labelling of substrates (TAILS) protocol had been developed in the Overall lab previously and we discussed using it on dental pulp tissue (Kleifeld et al., 2011a). Dr. Eckhard had removed one dental pulp from a tooth extracted by Dr. Mathew several months earlier, as a pilot project. The project began in May 2014. The first phase was collecting the pulp tissue. Dr. Mathew and I coordinated this that summer; I travelled to his office, where he extracted the donor wisdom teeth. I developed the technique for removing the pulp tissue from these teeth and freezing them on dry ice immediately. Together, Dr. Mathew and I collected all pulps studied in this research and in our previous study: he extracted the teeth, I then removed the tooth pulp tissue and froze it in GuHCl buffer. We obtained pulp tissue from a total of 16 donors over the course of the summer. Dr. U. Eckhard and I worked closely together that summer. We did all experiments together for donors 1-10 of 16 total. Dr. Eckhard worked very closely with me for the first run of the experiment (donors 1 and 2) and for the remainder of the summer I did all the experiments for donors 3-10 with Dr. Eckhard close-by for clarification and consultation. I was responsible for planning and implementing the histological component of this research, preparing a pulp, sectioning it, and staining it. We had other sections done by the UBC Wax-it service. I did all histology under the guidance of Dr. G. Tharmarajah. During the summer of 2015 I did initial analysis of LC-MS/MS data for all 16 pulp donors. I prepared spreadsheets of the data for further software analysis by Dr. Eckhard. iv At this point, Dr. Overall decided to divide the project in two. For donors 1, 2 and 11-16 Drs. Eckhard and G. Marino performed all further experiments to process the pulp proteins for LC-MS/MS analysis, did the analysis of these spectra, and prepared the figures for the first paper published from this data (Eckhard et al., 2015). For this first paper, I removed and did initial work on all pulp tissue used, and performed approximately 20 % of the experiments to process this tissue through PreTAILS and TAILS workflows. I also performed initial data analysis using Excel, reviewed the final manuscript, made edits and consulted from an endodontic perspective. I was also responsible for the histology image used in this paper. Through 2015 and 2016, I performed detailed software analysis on my LC-MS/MS data from donors 3-10, samples for which I had done the experimental work and analysis. In performing the analysis of MS spectra I worked closely with Dr. U. Eckhard and he did the final analysis to prepare our data for publication. I took the 5 day long Trans-Proteomic Pipeline introductory course given by the Seattle Institute of Systems Biology in the fall of 2015. Following this, I understand and can use all the software we employed to analyze our LC- MS/MS data, but my understanding of proteomics is not at the level of Dr. Eckhard and he was critical to final preparation of my data. In 2016 and 2017 I organized my data for publication, prepared the figures, and wrote the manuscript for a second paper that has been submitted to the Journal of Dental Research. Myself and Dr. Eckard share first author status on this paper. I presented new research posters, each serving as a landmark for my progress in this project, in every year of my program, January 2015, 2016, and 2017. At this year’s research day poster competition I received the M.Sc. student award. Our research project received ethical approval from the UBC Clinical Research Ethics Board (certificate # H13-03006). v Table of Contents Abstract ........................................................................................................................................ ii Lay Summary ............................................................................................................................. iii Preface ......................................................................................................................................... iv Table of Contents ...................................................................................................................... vi List of Tables ................................................................................................................................x List of Figures ............................................................................................................................ xi List of Abbreviations ............................................................................................................... xii Acknowledgements ................................................................................................................ xiii Dedication ................................................................................................................................. xiv Chapter 1: Introduction .............................................................................................................1 1.1 Basic science background to project ........................................................................ 1 1.1.1 Proteomics............................................................................................................. 1 1.1.2 Transcriptomics .................................................................................................... 1 1.1.3 Post-translational protein modifications ............................................................ 2 1.1.4 Protein variability with time ................................................................................. 2 1.1.5 The Human Proteome Project ............................................................................ 3 1.2 Dental pulp .................................................................................................................... 3 1.2.1 Tooth structure ...................................................................................................... 3 1.2.2 Odontoblast function within the pulp organ .....................................................