MASS SPECTROMETRY INTERFACED WITH ION MOBILITY OR LIQUID CHROMATOGRAPHY SEPARATION FOR THE ANALYSIS OF COMPLEX MIXTURES A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Danijela Smiljanic December, 2011 MASS SPECTROMETRY INTERFACED WITH ION MOBILITY OR LIQUID CHROMATOGRAPHY SEPARATION FOR THE ANALYSIS OF COMPLEX MIXTURES Dissertation Danijela Smiljanic Approved: Accepted: ________________________________ _________________________________ Advisor Department Chair Dr. Chrys Wesdemiotis Dr. Kim C. Calvo ________________________________ _________________________________ Committee Member Dean of the College Dr. Bi-min Zhang Newby Dr. Chand K. Midha ________________________________ _________________________________ Committee Member Dean of the Graduate School Dr. David Perry Dr. George R. Newkome ________________________________ _________________________________ Committee Member Date Dr. Yi Pang ________________________________ Committee Member Dr. Peter Rinaldi ii ABSTRACT This dissertation focuses on coupling separation techniques such as ion mobility (IM) and liquid chromatography (LC) to mass spectrometry and their application to characterization of complex mixtures. Non-covalent complexes between poly(ethylene imine) (PEI) and single stranded oligodeoxynucleotides (ODNs), as well as components from black raspberries, were characterized utilizing ion mobility mass spectrometry (IM- MS) and liquid chromatography mass spectrometry (LC-MS), respectively. Interfacing these separation methods to mass spectrometry allows for detection and identification of isobaric species and species present in low concentration. Non-covalent complexes between low molecular weight poly(ethylene imine) (PEI 400 and 800) and single-stranded oligodeoxynucleotides (ODNs) were investigated for five ODNs, including d(TTTTT), d(CCCCC), d(AAAAA), d(GGGGG) and d(GCGAT). In chapter 4 the compositions, as well as solution and gas-phase stabilities of the complexes (termed polyplexes) were examined by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS2). Independent of the mixing ratio of the reactants, the polyplex with 1:1 polymer-to-nucleotide stoichiometry, PN, is the dominant product. The gas-phase stabilities, assessed by MS2 and collisionally activated dissociation, follow the same order, providing evidence that the polyplex structures in aqueous solution and the more hydrophobic environment of the gas phase iii are very similar. Non-covalent complexes with different composition but the same molecular mass were corroborated by ion mobility mass spectrometry (IM-MS). In chapter 5 of this dissertation an investigation of the expanded non-covalent system, ternary complexes, of poly(ethylene imine), single-stranded oligodeoxynucleotides and glutamic acid entities, is discussed. The solution stabilities and gas-phase stabilities of the ternary complexes (termed terplexes) were examined by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS2). In addition, higher order ternary structures with multiple units of polymer and/or nucleotide present within the terplex were identified utilizing IM-MS. Finally, chapter 6 provides information on characterization of non-anthocyanin components from black raspberry fractions by interfacing liquid chromatography (LC) to mass spectrometry. The black raspberry extracts were provided from the lab of Dr. Joseph C. Scheerens at Ohio Agricultural Research and Development Center, Wooster, Ohio. Combination of LC-MS, ESI-MS and MS2 provided structural information for the corresponding components. iv DEDICATION To my parents Ana and Milutin Smiljanic and my brother Darijo Smiljanic, your love, support and sacrifice made this possible, and to my husband Josip Jovicic whose unconditional love and support helped me through the hard times. v ACKNOWLEDGEMENTS I would like to thank Dr. Wesdemiotis for his guidance, support and patience during my studies. I would also like to thank my committee members Dr. Bi-min Zhang Newby, Dr. David Perry, Dr. Yi Pang and Dr. Peter Rinaldi for their patience and helpful suggestions. I especially want to thank my parents Ana and Milutin Smiljanic, my brother Darijo and my husband Josip for always believing in me. I would like to thank all the past and current group members Alyson Leigh, Dr. Kittisak Chaicharoen, Dr. Sara Whitson, Dr. David Dabney, Dr. Bethany Subel, Madalis Casiano, AleerYol, Vincenszo Scionti, Bryan Katzenmayer, Shi Chunxiao, Kai Guo, Nhu Quynh Nguyen, Xiumin Liu, Nadrah Alawani and Dr. Xiaopeng Li for helpful discussions and friendships. Thank you for making the lab such a fun place. I would like to thank my friend Madalis Casiano (Daly) for always making me laugh, and for making the days in the lab, that were not so great, more enjoyable. I would also like to thank Liladher Pauldel for helpful discussions. vi TABLE OF CONTENTS Page LIST OF TABLES ...............................................................................................................x LIST OF FIGURES ........................................................................................................... xi CHAPTER I. INTRODUCTION ......................................................................................................1 II. INSTRUMENTAL METHODS BACKGROUND .................................................13 2.1 Mass Spectrometry ......................................................................................13 2.2 Ionization Methods ......................................................................................14 2.2.1 Electrospray Ionization (ESI)................................................................15 2.3 Mass Analyzers ............................................................................................19 2.3.1 Quadrupole Mass Analyzer ...................................................................20 2.3.2 Time-of-flight Mass Analyzer ..............................................................23 2.3.3 Quadrupole Ion Trap Mass Analyzer ....................................................26 2.3.4 Quadrupole Time-of-Flight (Q/TOF) Mass Analyzer ..........................30 2.4 Detectors ......................................................................................................31 2.5 Ion Mobility Mass Spectrometry (IMMS) ...................................................32 2.6 Liquid Chromatography Mass Spectrometry ...............................................37 2.6.1 Column and Separation Efficiency .......................................................39 III. MATERIALS AND INSTRUMENTATION ..........................................................44 vii 3.1 Materials ......................................................................................................44 3.2 Instrumentation .............................................................................................45 IV. NON-COVALENT COMPLEXES BETWEEN POLY(ETHYLENE IMINE) ANDSINGLE STRANDED DEOXYNUCLEOTIDES ..........................................49 4.1 Introduction ..................................................................................................49 4.2 Experimental Methods .................................................................................50 4.2.1 Mass Spectrometry Experiments ..........................................................50 4.3 Polyplexes with PEI 400 ..............................................................................52 4.4 Comparison of PEI 400 vs. PEI 800 ............................................................57 4.5 Intrinsic Stability of Polyplexes ...................................................................60 4.6 Ion Mobility Separation of the Polyplexes ..................................................64 4.7 Conclusions ..................................................................................................67 V. TERNARY COMPLEXES OF POLY(ETHYLENE IMINE), SINGLE STRANDED OLIGONUCLEOTIDES AND GLUTAMIC ACID MOIETIES ......69 5.1 Introduction ..................................................................................................69 5.2 Materials and Methods.................................................................................70 5.2.1 Mass Spectrometry Experiments ..........................................................71 5.3 Terplexes with PEI400.................................................................................72 5.4 C/N Ratio Effect ..........................................................................................84 5.5 Tandem Mass Spectrometry Studies on the Terplexes ................................88 5.6 Ion Mobility Separation of the Terplexes ....................................................90 5.7 Conclusions ..................................................................................................95 VI. CHARACTERIZATION OF PHENOLIC COMPOUNDS IN BLACK RASPBERRIES BY LIQUID CHROMATOGRAPHY MASS SPECTROMETRY (LC-MS) ...................................................................................96 viii 6.1 Introduction ..................................................................................................96 6.2 Sample Preparation ......................................................................................98 6.3 Instrumental Conditions ...............................................................................98 6.4 Characterization of Phenolic