Investigations into O-GlcNAcylation through analytical mass spectrometry by Thomas J. Clark B.Sc. (Physics), UNBC, 1997 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Chemistry Faculty of Science Thomas J. Clark 2014 SIMON FRASER UNIVERSITY Fall 2014 Approval Name: Thomas J. Clark Degree: Master of Science (Chemistry). Title: Investigations into O-GlcNAcylation through analytical mass spectrometry Examining Committee: Chair: Hua-Zhong Yu Professor David Vocadlo Senior Supervisor Professor George R. Agnes Supervisor Professor Andrew J. Bennet Supervisor Professor Bingyun Sun Internal Examiner Assistant Professor Date Defended/Approved: December 17, 2014 ii Partial Copyright Licence iii Abstract The modification of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is found on many proteins in the nucleus and cytoplasm. O-GlcNAc has been implicated in many physiological processes but much remains to be learned about the effects of this modification on protein function. In this thesis I detail two studies aimed to improve our understanding of protein modification by O-GlcNAc. First, I describe a bioinformatics study focused on uncovering the protein structural features that favour O-GlcNAcylation by the enzyme O-GlcNAc transferase. The search for a sequence or structural motif to be found amongst the many substrates O- GlcNAcylated by OGT is a path well-trodden. On the basis of our analysis of site mapping data accumulated from the literature and also through targeted site mapping of an entirely intrinsically disordered protein by mass spectrometry, I counter argue that OGT preferentially targets substrates which can be intrinsically disordered. Second, I describe a study aimed to gain insight into cellular proteomic response due to lowering of O-GlcNAc levels. There is mounting evidence O-GlcNAcylation is both linked with protein folding and intracellular proteome stability. While stability is measured by turnover it is also tied to misfolding. We investigated a possible relationship between lowered O-GlcNAc levels and instability of a segment of the proteome using stable isotope labelling by amino acids in cell culture mass (SILAC). This data reveals a change in protein stability between the entire sample populations that differ in O-GlcNAc levels, which is also observed for many individual proteins. Keywords: mass spectrometry; O-GlcNAc; N-acetylglucosamine transferase (OGT); site mapping; SILAC; protein stability iv Dedication Rosslyn, Ross and Julie. v Acknowledgements I would like to thank my supervisor Professor David J. Vocadlo for his patience, insight and provision of opportunities to learn and engage in novel research. I would also like to thank Professor Andrew J. Bennet and Professor George R. Agnes for their valuable recommendations as part of my thesis committee. I would like to thank Professor Christoph Borchers for his leadership in the BCPN and making his mass spectrometry resources available. I would like to thank the following mass spectrometry experts for brief but valuable discussions on mass spectrometry: Dr. Jun Han, Professor Pierre Thibault and Professor Leonard Foster. I thank Professor Pierre Thibault for the opportunity to perform research in his laboratory. I thank Dr. Yanping Zhu for his help wth Cell culture experiments and Dr. Scott Yuwa for recombinant tau protein. I would like to thank Darryl Hardie and Derek Smith for their tireless support of my mass spectrometry endeavours. Also, the following people made positive contributions to my completing this thesis: Angela Jackson, Suzanne Perry, Dr. Wesley Zandberg, Dr.Garrett Whitworth, Dr. Ta-Wei Lui, Dr. Xiaoyang Shan, Julia Heinonen, Tyra Cross, Dr. Scott Yuzwa and Isaac Seo and Dr. Samy Cecioni. vi Table of Contents Approval .......................................................................................................................... ii Partial Copyright Licence ............................................................................................... iii Abstract .......................................................................................................................... iv Dedication ....................................................................................................................... v Acknowledgements ........................................................................................................ vi Table of Contents .......................................................................................................... vii List of Figures.................................................................................................................. x List of Abbreviations and Acronyms .............................................................................. xv Chapter 1. Introduction ............................................................................................. 1 Historical introduction ...................................................................................................... 1 O-GlcNAcylation .............................................................................................................. 3 Mass spectrometry .......................................................................................................... 5 Chapter 2. Analysis of O-GlcNAc mapped sites reveals OGT targets regions of intrinsic disorder and unstable structures ........................ 13 Introduction ................................................................................................................... 13 Intrinsic disorder and the absence of structural data ............................................. 14 Sites mapped on an entirely intrinsic disorder protein ........................................... 19 Discussion ..................................................................................................................... 28 Methods ........................................................................................................................ 32 Data mining from literature ................................................................................... 32 Phyre2 Protein Homology/analogY Recognition Engine ....................................... 33 Site mapping Tau ................................................................................................. 34 Chapter conclusions and future speculation .................................................................. 34 Chapter 3. Proteome response to the removal and inhibition of OGT ................. 36 Introduction ................................................................................................................... 36 Experimental design ............................................................................................. 38 Results .......................................................................................................................... 42 Global comparison of population means for protein turnover between 5S- GlcNAc treated and untreated HEK293 cells. ............................................... 42 Differentiated protein synthesis measured by heavy to light isotope ratios due to reduced O-GlcNAcylation .................................................................. 47 Differentiated protein degradation measured by medium to light isotope ratios due to reduced O-GlcNAcylation ........................................................ 50 Differentiated protein degradation measured by medium to light isotope ratios due to reduced O-GlcNAcylation ........................................................ 51 Proteins with differentiated synthesis conserved across experiments due to reduced levels of O-GlcNAcylation ............................................................... 53 Results surrounding a question of whether reduced O-GlcNAcylation levels alters the turnover of chaperone proteins ..................................................... 55 Time dependent kurtosis of H/M turnover data ..................................................... 67 vii Discussion ..................................................................................................................... 69 Discussion or results ............................................................................................ 69 Discussion of the rejection of MEF medium to light data sets. .............................. 71 False positives in preliminary MEF cell experiment traced to missed cleavages and high background ................................................................... 75 Conversion of Arginine to Proline in SILAC Experiments ...................................... 77 Inhibition of over alkylation by Iodoacetaminic acid ............................................... 78 Improving proteome coverage through comparison of fragmentation mechanisms ................................................................................................. 79 Methods ........................................................................................................................ 81 Cell Culture ........................................................................................................... 81 SILAC Labelling .................................................................................................... 82 SILAC experiment with MEF cells ......................................................................... 83 SILAC experiment with HEK293 cells ..................................................................