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Front-End Methods for Enhancing the Analytical Power of Mass Spectrometry

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Front-End Methods for Enhancing the Analytical Power of Mass Spectrometry FRONT-END METHODS FOR ENHANCING THE ANALYTICAL POWER OF MASS SPECTROMETRY PETER PAUL LIUNI A DISSERTAITON SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FUFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN CHEMISTRY YORK UNIVERSITY TORONTO, ONTARIO December 2015 © Peter Paul Liuni, 2015 Abstract The analytical power and versatility of mass spectrometry can be enhanced by adding ‘front-end’ devices, which provide additional functionality before, during or immediately after ElectroSpray Ionization (ESI). Such devices can include Ion mobility spectrometry (IMS) and Time-Resolved ElectroSpray Ionization (TRESI) which provide enhanced analysis of illicit compounds, protein folding, enzyme kinetics, and catalysis-linked dynamics. With respect to IMS, this work describes implementation of a hybrid Trace Compound Detector (TCD) system that combines IMS and MS to allow for rapid front- end mobility separation, followed by characterization and identification of analytical markers of seized opium by mass spectrometry. Ultimately, this device provides an avenue for rapid prosecution based on simultaneous detection and unambiguous identification of illicit drugs. TRESI is used to extend Mass Spectrometry (MS) to millisecond-timescale reaction studies. In the first instance, we combine TRESI with Travelling Wave Ion Mobility Spectrometry (TWIMS) to compare equilibrium and kinetic unfolding intermediates of cytochrome c, showing a high degree of correlation between all species populated under these substantially different regimes. We then combine TRESI with Hydrogen Deuterium Exchange (TRESI-HDX) to elucidate the relationship between structural fluctuations (conformational dynamics) of enzymes and their catalytic activity. The results of this work include a new model for catalysis-linked dynamics, in which the nature of the conformational landscape explored by an enzyme is independent of catalysis, but the rate at which the landscape is explored is enhanced for catalytically active species. ii Acknowledgements You know, I have a confession to make. Keep your hearts in your chests because this data’s all real. But really, a true confession. And that is, I was a failing high school chemistry student. Now, if it had been up to my guidance counselors, parents, friends, and family; my journey would have ended back in high school. Full stop. But the question that remained inside me was why? Why if you are so passionate about a subject, so vividly enthralled by everything about it, why should you be told you can’t continue to learn it? So I enrolled in College. No knock to College, but when all your friends and family sail off to University – there’s this nagging voice inside your head that wants to keep telling you one thing. You are lesser. And maybe I needed to be told that, because my 1st year of College was humbling to say the least – I failed Chemistry yet again. The voice was right, or so it seemed. Sometime during the winter of 2003 I came to realise I was a poor student; something I never admitted to myself. It sank in deep, and what erupted was outright rage. How could I let MY dream, MY goal, just slip away? How could I lie to my 4 year old self, who drew a picture of a Scientist, proclaiming that is what he wanted to be when he grew up on his Senior Kindergarten Graduation Day? That thought right there, changed me forever. I channeled it into pure blind determination. And I studied. Harder than I ever had in my life. I would never accept failure again, and I’d go even further, I wouldn’t accept it in my closest friends. I studied hard for them too, because I knew they had dreams. And no one should ever be told by anyone – even themselves – to give up on their dreams. iii The quest for knowledge that is a Ph.D. is a long journey for the most part made in solitude, but also in companionship. The names here represent people who have enriched and made a meaningful - however large or small - contribution to my life from start to end of this Ph.D. Teresa and Pietro Liuni, my parents, thank you for your unwavering support and love throughout my long 12 years of post-secondary education, for every step I thought I took alone, you were behind me, ready to catch me if I fell. To my Family, Pupo and Liuni, you are my backbone and my blood, thank you for being there for me and reminding me what family is all about. Carmine and Jeanine, my best friends, you have always been there for me, a ray of light for when I was in the darkest of places, even when all other lights had gone out. Derek Wilson, my advisor and mentor, thank-you for letting me play in your lab. The creative space you gave me made me feel like a child again; free to create and explore in my ideas. To my Committee members, Dasantila Golemi-Kotra, Sergey Krylov, Gerald Audette, Vivian Saridakis, and Grary Shaw; your valuable wisdom and suggestions have made this dissertation possible. John Van Nostrand, for teaching me mass spectrometry and for being a good friend. Greg Koyanagi, for your ability fix nearly everything and being a good friend. Diethard Bohme, for your essential advice and criticisms of my work. Vlad Romanov, for teaching me how to be confident. Voislav Blagojevic, for giving me hope that this whole thing’s going to eventually pan out. Tamanna Rob, for being the lab mom, my confidant and friend. Yanfang Liang, for always making it sound easy. Declan Williams, for showing me true work ethic, and Resistance. Shaolong Zhu, for being the better version of me. Diana iv Resetca, for teaching me work ethic and vodka can definitely mix. Preet Gill, for those late night talks bro. Cristina Lento, for being the only Italian in the lab – relate. Kereene Brown, for being an upbeat presence and the Catan box. Bin Deng, for your versatility and modesty. Diefei Sun, for your useful discussions and help. Marlee Ng, for making everything memorable and being a great friend. Karan Hingorani, for your inspiration and love of science and beer. I leave this poem as a final ode to my life as a student, it has given me strength and inspiration; my hope is that it does the same for you. All that is gold does not glitter, Not all those who wander are lost; The old that is strong does not wither, Deep roots are not reached by the frost. From the ashes a fire shall be woken, A light from the shadows shall spring; Renewed shall be blade that was broken, The crownless again shall be king. -J.R.R Tolkein v List of Publications 1. Liuni, P.; Deng, B.; Wilson, D. J. Comparing Equilibrium and Kinetic Protein Unfolding Using Time-Resolved Electrospray-Coupled Ion Mobility Mass Spectrometry. Analyst 2015, 140 (20), 6973–6979. 2. Liuni, P.; Romanov, V.; Binette, M.-J.; Zaknoun, H.; Tam, M.; Pilon, P.; Hendrikse, J.; Wilson, D. J. Unambiguous Characterization of Analytical Markers in Complex, Seized Opiate Samples Using an Enhanced Ion Mobility Trace Detector-Mass Spectrometer. Anal. Chem. 2014, 86 (21), 10772–10779. 3. Liuni, P.; Zhu, S.; Wilson, D. J. Oxidative Protein Labeling with Analysis by Mass Spectrometry for the Study of Structure, Folding, and Dynamics. Antioxid. Redox Signal. 2014, 21 (3), 497–510. 4. Liuni, P.; Olkhov-Mitsel, E.; Orellana, A.; Wilson, D. J. Measuring Kinetic Isotope Effects in Enzyme Reactions Using Time-Resolved Electrospray Mass Spectrometry. Anal Chem 2013, 85 (7), 3758–3764. 5. Liuni, P.; Jeganathan, A.; Wilson, D. J. Conformer Selection and Intensified Dynamics During Catalytic Turnover in Chymotrypsin. Angew. Chem. 2012, 124 (38), 9804–9807. 6. Rob, T.; Liuni, P.;Gill, P.K.; Zhu, S.; Balachandran, N.; Berti, P.J.; Wilson D.J. Measuring Dynamics in Weakly Structured Regions of Proteins Using Microfluidics-Enabled Subsecond H/D Exchange Mass Spectrometry. Anal. Chem. 2012, 84 (8), 3771-3779. 7. Liuni, P.; Wilson, D. J. Understanding and Optimizing Electrospray Ionization Techniques for Proteomic Analysis. Expert Rev. Proteomics 2011, 8 (2), 197– 209. 8. Liuni, P.; Rob, T.; Wilson, D. J. A Microfluidic Reactor for Rapid, Low-Pressure Proteolysis with on-Chip Electrospray Ionization. Rapid Commun. Mass Spectrom. 2010, 24 (3), 315–320. vi Table of Contents Abstract.............. ................................................................................................................ ii Acknowledgements .......................................................................................................... iii List of Publications .......................................................................................................... vi Table of Contents ............................................................................................................ vii Abbreviations.... ............................................................................................................... xi List of Tables....................................................................................................................xii List of Figures.... ............................................................................................................. xiii Chapter 1: Introduction ................................................................................................... 1 1.1 Mass Spectrometry.............................................................................................. 2 1.1.1 Current Perspectives ....................................................................................... 2 1.1.2 Soft Ionization Methods .................................................................................
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