A Thesis entitled Elucidating the Sequence and Structural Features of Human Bence-Jones Proteins by Weliwaththage Thilini Perera Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science in Chemistry _______________________________________ Dragan Isailovic, Committee Chair _______________________________________ Dr. Leif Hanson, Committee Member _______________________________________ Dr. John Bellizzi, Committee Member ______________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2018 Copyright 2018, Weliwaththage Thilini Perera This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Elucidating the Sequence and Structural Features of Human Bence-Jones Proteins by Weliwaththage Thilini Perera Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Chemistry The University of Toledo August 2018 Amyloidosis diseases are characterized by the deposition of insoluble protein aggregates called amyloids. More than 30 diseases are associated with amyloid-forming proteins, including Alzheimer’s, Parkinson’s, Huntington’s and immunoglobulin light chain amyloidosis (AL amyloidosis). The proteins contributing to each disease have distinct primary structures. The mechanism of amyloid formation is not well understood, but appears to be associated with protein misfolding processes accompanied by self- aggregation. In AL amyloidosis, immunoglobulin light chain proteins become misfolded and accumulate as amyloid. No effective therapeutic solution is available to treat this disease condition, which is usually fatal a few months after diagnosis. AL amyloidosis appears in a subset of patients with multiple myeloma (MM), a malignant disease condition characterized by bone marrow failure. Most of the patients having MM excrete monoclonal free immunoglobulin light chains, also called Bence-Jones proteins, into the urine. In contrast to AL amyloidosis, amyloid deposits are not observed in vivo in most patients suffering from MM. Consequently, the identification of the precise sequence and structural i information of proteins related to MM and AL amyloidosis are essential to understand the factors responsible for aggregation. As reported in the literature, several analytical methods have been used to study primary, secondary, tertiary and quaternary structures of amyloid-forming proteins. In our study, mass spectrometry, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) were used to obtain sequence and structural information about immunoglobulin light chain proteins, isolated from urine of AL amyloidosis and MM patients. Accordingly, molecular masses of seven Bence-Jones protein samples were measured by ESI-MS and MALDI-MS and the properties of their ions in the gas phase were observed by IMS-MS. Since five of them are with unknown sequences, the bottom- up proteomic approach was used to obtain unreported sequences of immunoglobulin light chain proteins. The enzyme-digested protein samples were analyzed by MALDI-MS, ESI- MS, IMS-MS and HPLC-ESI-MS/MS. A de novo sequencing-assisted database search was performed using PEAKS search tool to obtain possible peptide sequences. Multistep MS/MS approaches were applied to differentiate between leucine and isoleucine residues present in newly identified peptides. Furthermore, CD spectroscopy was used to compare the secondary structure elements of different light chain proteins. Most light chains showed high percentage of β-sheets, which is common for amyloid-forming proteins. In addition, DLS was used to study the effect of physiochemical parameters on the aggregation behavior of light chain proteins. Overall, novel sequence and structural features of ii immunoglobulin light chains were obtained by these approaches and will be correlated with the properties of other amyloid-forming proteins. iii Acknowledgements First and foremost, I would like to thank my advisor, Dr. Dragan Isailovic, for his immense support, motivation and valuable time sent on me for the discussions form initial to final stage of this project. I would also like to thank Dr. Leif Hanson, for providing me biological samples and the constant support at all stages including the guidance to conduct MALDI and DLS experiments. I express my gratitude to Dr. Bellizzi, Dr. Mueser and Dr. Edmundson for their valuable advices and suggestions. I would like to convey my special thanks to Dr. Erickson for his assistance to perform CD spectroscopy experiments. Many thanks to all current and former lab members in Isailovic’s lab for being so nice and helpful throughout and making lab such a wonderful place to work in. This includes Krishani, Sanjee, David, Rachel, Siddhita, Jen and Kevin. My special thanks go to Dr. Rachel Marvin for her continuous help to solve scientific problems since the beginning of my research work. Most importantly, I owe my deepest gratitude to my loving family including my husband, parents and siblings for constant support. Their unconditional love and continuous encouragement made me confident to go through all challenging situations in graduate life at University of Toledo. iv Table of Contents Abstract ................................................................................................................................ i Acknowledgements ............................................................................................................ iv Table of Contents .................................................................................................................v List of Tables .................................................................................................................... ix List of Figures .................................................................................................................... xi List of Abbreviations .........................................................................................................xv 1Introduction … .................................................................................................................. 1 1.1 Amyloidosis Diseases .......................................................................................... 1 1.2 Immunoglobulin Light Chain Amyloidosis (AL amyloidosis) ............................ 3 1.3 Analytical Methods Used to Study Amyloid Protein Structure, Folding and Assembly.............................................................................................................. 5 1.4 Mass Spectrometry ............................................................................................... 6 1.5 Ion Sources in Mass Spectrometry ....................................................................... 7 1.5.1 Matrix-Assisted Laser Desorption/Ionization (MALDI) .............................. 8 1.5.2 Electrospray Ionization (ESI) ..................................................................... 10 1.6 Mass Analyzers .................................................................................................. 12 v 1.6.1 Quadrupole Mass Analyzer......................................................................... 13 1.6.2 TOF Mass Analyzer .................................................................................... 15 1.6.3 Ion Trap Mass Analyzer.............................................................................. 17 1.6.4 Orbitrap Mass Analyzer .............................................................................. 19 1.7 Proteins and Proteomics ..................................................................................... 20 1.7.1 Protein separation by gel electrophoresis ................................................... 21 1.7.2 Protein digestion ......................................................................................... 22 1.8 Mass Spectrometry-Based Proteomics ............................................................... 22 1.8.1 Tandem Mass Spectrometry (MS/MS) ....................................................... 24 1.8.2 Peptide Fragmentation ................................................................................ 25 1.8.3 Peptide Mass Fingerprinting (PMF) ........................................................... 26 1.8.4 Protein Sequencing Techniques .................................................................. 27 1.8.5 De novo Sequencing of Peptides ................................................................ 28 1.9 MS-based leucine (Leu) and isoleucine (Ile) discrimination approach .............. 30 1.10 Mass Spectrometry-Chromatography Coupling................................................. 32 1.11 Ion mobility spectrometry (IMS) ....................................................................... 33 1.12 Sequencing of immunoglobulin light chains by MS .......................................... 34 1.13 Circular Dichroism (CD) Spectroscopy ............................................................. 35 vi 1.14 Dynamic light scattering (DLS) ......................................................................... 36 2 Materials and methodology................................................................................ 38 2.1 Materials and instruments .................................................................................. 38 2.2 Methodology .....................................................................................................