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The Characterization of Cell Surface Receptor Complexes by Affinity Chromatography, Liquid Chromatography and Tandem Mass Spectrometry

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The Characterization of Cell Surface Receptor Complexes by Affinity Chromatography, Liquid Chromatography and Tandem Mass Spectrometry The Characterization of Cell Surface Receptor Complexes by Affinity Chromatography, Liquid Chromatography and Tandem Mass Spectrometry by Jaimie Dufresne BSc, MSc, Ryerson University A dissertation presented to Ryerson University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Program of Molecular Science Toronto, Ontario, Canada, 2017 © Jaimie Dufresne 2017 AUTHOR'S DECLARATION FOR ELECTRONIC SUBMISSION OF A DISSERTATION I hereby declare that I am the sole author of this dissertation. This is a true copy of the dissertation, including any required final revisions, as accepted by my examiners. I authorize Ryerson University to lend this dissertation to other institutions or individuals for the purpose of scholarly research. I further authorize Ryerson University to reproduce this dissertation by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. I understand that my dissertation may be made electronically available to the public. ii Abstract Cell surface receptors are of critical importance to the treatment of disease but are difficult to isolate and identify by classical approaches. Here, a robust and general method for capturing a receptor complex from the surface of live cells with ligands presented on nanoscopic beads is demonstrated. Two forms of affinity chromatography: the presentation of a biotinylated ligand to the surface of live cells and recovered by classical affinity chromatography was compared to the presentation of the ligand on the surface of nanoscopic chromatography beads for the isolation of the IgG-FcR complex from the surface of live cells. The IgG ligand was first characterized by LC-ESI-MS/MS and compared to controls by classical statistical methods in order to minimize the total error rate of protein identification. The first strategy was to isolate the IgG-FcR complex using biotinylated IgG (IgG-B) compared to IgG with a DTT- cleavable biotin spacer arm (IgG-SS-B) to activate and capture the receptor associated supramolecular complex. In this method, live cells were incubated on ice with IgG, IgG-B or IgG-S-S-B, for 30 minutes, washed and disrupted by French press. After washing, the IgG-B and IgG-S-S-B ligands were eluted from a streptavidin-agarose column in DTT followed by mercaptoethanol, 2M NH4OH and then 50mM biotin in 2 M NH4OH and the ligand remaining on the columns was scrubbed with 2% SDS or digested with trypsin. The analysis of the eluted fractions and SDS scrub by dot blots or Western blots with streptavidin-HRP (SA-HRP) or donkey anti human IgG-HRP (Dk anti-hIgG-HRP) probes agreed with liquid chromatography and tandem mass spectrometry (LC-ESI-MS/MS) of the tryptic peptides that IgG-B or IgG-S-S- B was non-specifically eluted by ammonium hydroxide buffer. LC-ESI-MS/MS of the DTT eluted fraction from the charged streptavidin column (agarose-SA-B-S-S-IgG) to release the S- IgG ligand showed detectable amounts of the S-IgG complex and minimal amounts of Fc and Fcrl proteins. Some of the known components of the Fc mediated phagocytic pathway were enriched in DDT and BME in the Biotin-S-S-IgG-FCGR compared to Biotin-IgG controls. Many variations of the classical ligand affinity chromatography method for the isolation of cell surface receptors were investigated including the use of detergents, heat aggregated ligands, the use of cross linkers and the capture of IgG over streptavidin or protein G chromatography. None of these methods specifically captured Fc receptors however the identified proteins were different from background binding controls In the second strategy, IgG coated PMMA and melamine micro and glass nano beads were presented to live cells, homogenized by French pressing, isolated by ultracentrifugation and then extracted by a salt and acetonitrile step gradient. Laser confocal scanning microscopy, dot blots with Coomassie staining or Western blot all confirm the three nano and micro bead surfaces: silica, melamine and PMMA were coated in IgG and were able to bind RAW 264.7 macrophages. Fc receptors were identified in significant numbers in the silica nano treatments compared to controls as well as members of the FcR mediated phagocytic pathway with very little background binding. In contrast to the strategy of classical affinity chromatography applied to integral membrane receptors, the use of ligand coated micro, and as shown here for the first time, silica nano beads, was effective at binding and capturing a receptor from the surface of a live cell and may serve as a general method for isolating receptor complexes on the nano scale. iii Acknowledgments For my beloved husband Daniel who makes life joyous and beautiful. For my sister Billie, and my mother and father Sylvie and Philip who have loved and supported me unconditionally. My heartfelt thanks to my cherished friends and lab mates who are like family to me: Elissa, Kathy, Sabrina, Eun Jee, Thanusi, Pardis, Bobbi, Harvey, John O., Erin, Shelby, Jamie, Lina, Dave, Leslie, Christina, Andy, Regan, Guy, Eric, Susan, Amr, Rasha, and Fiona. Thank you to the many students who have taken part in this work and put forth exceptional efforts: Gar-Way, Trung, Moumita, Eun Jee, Morla and Zhou. Thank you to Angelique and Peter for their mentorship and guidance. My deepest gratitude to my supervisor Dr. John Marshall for his support and encouragement. I will be forever grateful for the opportunity he gave me. iv Table of Contents Abstract .......................................................................................................................................... iii Table of Contents ............................................................................................................................ v List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii List of Abbreviations ................................................................................................................... xvi List of Abbreviations for experimental treatments .................................................................... xviii INTRODUCTION .......................................................................................................................... 1 MATERIALS AND METHODS .................................................................................................. 36 RESULTS ..................................................................................................................................... 56 DISCUSSION ............................................................................................................................. 133 CONCLUSION: .......................................................................................................................... 146 SUPPLEMENT A ....................................................................................................................... 160 Treatments and ESI-LC-MSMS runs for ligand affinity chromatography experiments ............ 161 SUPPLEMENT B ....................................................................................................................... 165 SQL Query Code Used for grouping FC and IgG Tables:.......................................................... 166 SUPPLEMENT C ....................................................................................................................... 175 R code for the groupings of Fc tables ......................................................................................... 176 SUPPLEMENT D ....................................................................................................................... 186 Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry ..................................................................................................................................................... 187 SUPPLEMENT E ....................................................................................................................... 231 Freeze-dried plasma proteins are stable at room temperature for at least one year .................... 232 SUPPLEMENT G ....................................................................................................................... 277 v The endogenous tryptic peptides of EDTA plasma from Alzheimer’s Dementia, Heart Attack, Sepsis, Ovarian Cancer, Breast Cancer, and Multiple Sclerosis and institution/study matched controls ........................................................................................................................................ 278 vi List of Tables Table I. The filtering of the cumulative, redundant correlations to protein accession sequences on the basis of charge state and peptide sequence. Bulk IgG, blank, dust and random spectra were searched against a federated human protein library. Myosin was searched against a federated rabbit library. The redundant use of MS/MS spectra to correlate to more than one peptide sequence was eliminated using a composite key in SQL Server. Table 2. Peptide counts of hIgG from biotinylated monovalent and polyvalent ligands applied to crude or live cells
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