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Downloaded from Uniprot (Release 2018 09; 20,410 Sequences) and Common Contaminants Were Embedded from Maxquant DEVELOPMENT OF HIGH-EFFICIENCY UNDECANAL-BASED N TERMINI ENRICHMENT (HUNTER) FOR MONITORING PROTEOLYTIC PROCESSING IN LIMITED SAMPLES by Shao Huan Samuel Weng B.Sc., Simon Fraser University, 2013 M.Sc., Simon Fraser University, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2019 © Shao Huan Samuel Weng, 2019 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: Development of High-efficiency Undecanal-based N Termini EnRichment (HUNTER) for Monitoring Proteolytic Processing in Limited Samples submitted by Shao Huan Samuel Weng in partial fulfillment of the requirements for the degree of Master of Science in Pathology and Laboratory Medicine Examining Committee: Dr. Philipp F. Lange, Pathology and Laboratory Medicine Supervisor Dr. Thibault Mayor, Biochemistry Supervisory Committee Member Dr. Mari DeMarco, Pathology and Laboratory Medicine Additional Examiner Dr. James Lim, Pediatrics Additional Examiner ii Abstract Genes encode the information for the amino acid backbone of proteins. This information can be altered by genetic variation or alternative splicing and alternative initiation of translation. After translation the protein can further alter by post-translational modification. All these different versions of a protein encoded by one gene are termed proteoforms. Protein N termini can be used to identify truncated (proteolytically cleaved), alternatively translated, or N terminally modified proteoforms that often have distinct functions. Cleavage of proteins by proteases is frequently altered in disease, including cancers and following the occurrence and loss of protein N termini can pinpoint abnormal proteolytic activity in disease. Selective enrichment of N-terminal peptides is necessary for proteome-wide coverage for unbiased identification of site-specific proteolytic processing and protease substrates; however, for comprehensive study of N termini so-called N-terminome analysis, most N termini enrichment techniques require relatively large amounts of starting material in the range of several hundred micrograms to milligrams of crude protein lysate. Due to sample constraints, this type of analysis cannot be routinely applied to clinical biopsies, especially those from pediatric patients. We present High-efficiency Undecanal-based N Termini EnRichment (HUNTER), a robust, sensitive, and scalable method for the analysis of previously inaccessible microscale samples. With this approach, >1,000 N termini are identified from a minimum of 2 µg raw HeLa cell lysate and >5,000 termini from 200 µg of raw HeLa lysate with high-pH pre-fractionation. We demonstrate the broad applicability of HUNTER with the first N-terminome analysis of sorted human primary immune cells and enriched mitochondrial fractions from pediatric cancer patients. The workflow was implemented on a liquid handling system to demonstrate the feasibility of automated liquid biopsy processing from pediatric cancer patients. In general, HUNTER method benefits in handling rare and precious clinical samples. iii Lay Summary The leading string of amino acids in a protein (i.e., the N-terminus) and can provide useful information about protein characteristics and functions. Abnormal cutting of proteins by deregulated enzyme called proteases, influences cellular regulation and communication in most cancers. Proteases constitute a major target for cancer drugs, and their breakdown activity can be monitored by following the occurrence and loss of protein termini. Therefore, there is a growing interest in identifying protein N termini and their modification; however, most termini selection methods require large amounts of raw material (>100 µg). Here, we developed a robust, sensitive, and automated method termed High-efficiency Undecanal-based N Termini EnRichment (HUNTER), for identification of thousands of N termini from limited sample amounts (>1 µg). HUNTER benefits in handling rare and precious clinical samples. We also have applied HUNTER to identify the N-terminal profiles of sorted immune cells, subcellular compartments, and plasma from childhood cancer patients. iv Preface The work on this dissertation was done under the guidance and mentorship from Dr. Philipp Lange. Janice Tsui assisted me with HUNTER automation. Lorenz Nierves and Janice Tsui helped me with automated N termini analysis in patient blood plasma (BP) and bone marrow interstitial fluid (BM). Dr. Anuli Uzozie and Lorenz Nierves designed, performed, and analyzed mitochondria experiments. Dr. Anuli Uzozie assisted with optimizing, performing, and analyzing data-independent acquisition experiments. Dr. Fatih Demir conducted rat brain and Arabidopsis leaf experiments. All data analysis was completed with assistance from Enes Ergin. I carried out all other experiments, data analysis, and data interpretation shown in this thesis, in collaboration with Dr. Pitter F. Huesgen and Dr. Fatih Demir. Chapter 2 and 3 are reproduced in part from Weng et al. Sensitive determination of proteolytic proteoforms in limited microscale proteome samples, Mol. Cell. Proteom., doi:10.1074/ mcp.TIR119.001560 (2019). Primary pediatric B-cell acute lymphoblastic leukemia and acute myeloid leukemia patient mononuclear cells enriched from bone marrow aspirates, BP and BM were retrospectively sourced from the Biobank at BC Children’s Hospital following informed consent and approval by the University of British Columbia Children’s and Women’s Research Ethics Board (REB #H15- 01994) in agreement with the Declaration of Helsinki. Patient BP and BM samples were collected at the time of diagnosis and 29 days after induction chemotherapy. Peripheral blood mononuclear cells from healthy donors were obtained following informed consent and approval by the University of British Columbia Children’s and Women’s Research Ethics Board (REB #H10- 01954). v Table of Contents Abstract ........................................................................................................................ iii Lay Summary ............................................................................................................... iv Preface ........................................................................................................................... v Table of Contents ......................................................................................................... vi List of Figures ............................................................................................................... x List of Abbreviations .................................................................................................. xii Acknowledgements .................................................................................................... xv Dedication .................................................................................................................. xvi Chapter 1 Introduction ............................................................................................. 1 1.1 General introduction ............................................................................................... 1 1.1.1 Proteoforms ..................................................................................................... 1 1.1.2 Post-translational modifications..................................................................... 2 1.1.2 Proteases and proteolysis ............................................................................. 3 1.1.3 Proteases in cancer development ................................................................. 4 1.2 Current N termini enrichment methods: from positive to negative selection ............ 6 1.2.2 Positive selection N termini enrichment methods .......................................... 7 1.2.3 Negative selection N termini enrichment methods ...................................... 10 1.3 Objective .............................................................................................................. 22 vi Chapter 2 Development of High-efficiency Undecanal-based N Termini EnRichment (HUNTER) ......................................................................... 23 2.1 Introduction .......................................................................................................... 23 2.2 Method ................................................................................................................. 24 2.2.2 Materials and reagents ............................................................................... 24 2.2.2.1 HeLa cell line ........................................................................................... 24 2.2.2.2 Plant material .......................................................................................... 24 2.2.2.3 Rat brain samples ................................................................................... 24 2.2.3 Preparation of single-pot solid-phase-enhanced sample-preparation beads 25 2.2.4 Preparation of stop-and-go-extraction tips .................................................. 25 2.2.5 Fluorometric and colorimetric protein and peptide measurements .............. 25 2.2.6 High-pH reversed phase fractionation ......................................................... 25 2.2.7 Optimization of HUNTER
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