MASS SPECTROMETRY TECHNIQUES FOR THE CHARACTERIZATION OF SYNTHETIC POLYMERS, BIOPOLYMERS, BIODEGRADATION PRODUCTS AND THEIR INTERACTIONS A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Madalis Casiano-Maldonado May, 2012 MASS SPECTROMETRY TECHNIQUES FOR THE CHARACTERIZATION OF SYNTHETIC POLYMERS, BIOPOLYMERS, BIODEGRADATION PRODUCTS AND THEIR INTERACTIONS Madalis Casiano-Maldonado Dissertation Approved: Accepted: ________________________________ ________________________________ Advisor Department Chair Dr. Chrys Wesdemiotis Dr. Kim C. Calvo ________________________________ ________________________________ Committee Member Dean of the College Dr. Abraham Joy Dr. Chand K. Midha ________________________________ ________________________________ Committee Member Dean of the Graduate School Dr. David S. Perry Dr. George R. Newkome ________________________________ _______________________________ Committee Member Date Dr. Peter Rinaldi ________________________________ Committee Member Dr. Michael J. Taschner ii ABSTRACT The characterization of synthetic polymers and complex oligomeric mixtures by a combination of different mass spectrometry (MS) techniques is the main topic of this dissertation. Ion mobility (IM) separation and/or tandem mass spectrometry (MS2) were interfaced with single stage mass spectrometry for the fast, complete, precise and accurate characterization of different polymers, biopolymers, and biodegradation products. Additionally, the development of a mass spectrometry protocol for the quantitation of proteins adsorbed on different polymer surfaces was accomplished in this dissertation. Over the passed years, synthetic routes for the creation of PEGs have been modified by the elimination of organic catalysts and solvents and the application of green chemistry for the generation of new polymers. In Chapter 4 of this dissertation, MS and MS2 were employed, as the main analytical techniques, for the structural elucidation of enzyme- catalyzed, functionalized PEG and tetraethylene glycol (TEG) biomaterials. The samples analyzed were synthesized by two different processes, transesterification and Michael addition reactions, both of them using CALB as the catalyst. In Chapter 5 of this dissertation, polylactide was degraded using Proteinase K and its biodegradation products were analyzed by MS and MS2. Elucidation of its degradation products is important to the biomedical community; knowledge of these products helps to iii see any toxicity problems with the use of this polymer. The MS and MS2 results showed that the degradation products are short polylactide chains, down to the monomer. No other, potentially dangerous organics were detected. In the following chapter of this dissertation (Chapter 6), the adsorption of three model proteins onto two different surfaces was evaluated over a pH range. The results were explained on terms of polymer chemistry, surface morphology, proteins’ isoelectricpoints and molecular dimensions. Finally, Chapter 7 reports how derivatization, degradation, or chromatographic separation can be avoided for the analysis of complex systems, if multidimensional mass spectrometry methods combining ion mobility separation and different ionization techniques are employed. Using this new approach complex poly(α-peptoid) samples were characterized. The results showed that the main product in the system contained the N-heterocyclic carbene (NHC) moiety that served as ring-opening polymerization catalyst. Furthermore, it was demonstrated that elimination of the NHC segment leads to cyclic poly(α-peptoid)s that have very similar conformations with comparably sized polypeptides iv DEDICATION To my role model, whom I admire and love, my mom, Amada Maldonado-Nazario. To my dad and sisters, for your unconditional support, Jose Anibal Casiano, Jeannette M. Casiano, Lizbeth Casiano, Roxana Casiano, and Rosa M. Casiano. To my husband, who was my first motivator, supporter and my eternal love, Lyn Gabriel Muñoz-Robledo. To my latest inspiration, my daughter, Mikaela Sophia Muñoz-Casiano. v ACKNOWLEDGEMENTS First, I thank my God, you are The one who was my principal support. Thanks because you help me to keep myself motivated and focused on my dream. I wish to thank Dr. Chrys Wesdemiotis for his guidance, advice and support throughout this project. It was the most enrichment experience in my academic life and was a pleasure to work for him. I want to thanks Dr. Xiaopeng Li because I was fortunate to receive his help and advices. My special thanks to Dr. Goy Teck Lim, Dr. Judith E. Puskas and Kwang Su Seo for all the input, advice and for providing many sample which are the main part of this dissertation. Thanks to Li Guo and Donghui Zhang, who believed in my work and permitted me to collaborate with them. I would like to thank my committee members for actually take the time to read my dissertation and all their feedback: Dr. Abraham Joy, Dr. Michael J. Taschner, Dr. Peter Rinaldi, and Dr. David S. Perry. I would like to express my thankfulness to Nilufer Solak, Danijela Smiljanic- Jovicic, Gladys Rocio Montenegro Galindo, Tejal Deodhar, Saida Y. Garcia and Aleer Yol. Thanks for all the wonderful memories we built together during these years. Also, I vi wish to thanks Nilufer Solak, David E. Dabney, Wesdemiotis former and current research group for sharing with me their expertise in mass spectrometry. I would also like to thank the Department of Chemistry and the State of Ohio for providing financial support while conducting my graduate studies at the University of Akron. Thanks to all my Akron friends for their company. They made me feel at home. Finally, thanks to my family who believe in me, who gave me support almost every day, who were capable to hear complaints. Thanks for being the wonderful family ever: Amada Maldonado, Jose Anibal Casiano, Jeannette M. Casiano, Lizbeth Casiano, Roxana Casiano, Rosa M. Casiano, all my nieces and brothers in law. Thanks to my husband, Lyn G. Muñoz, for his trust, patience, help and love vii TABLE OF CONTENTS Page LIST OF TABLES.………………………………………………………………… xiii LIST OF FIGURES..……………………………………………………………….. xiv LIST OF SCHEMES.……………………………………………………………… xix LIST OF EQUATIONS…………………………………………………………..... xxi CHAPTER I. INTRODUCTION……………………………………………………………. 1 II. MASS SPECTROMETRY BACKGROUND……………………………….. 8 2.1 Mass Spectrometry…………………………………………………. 8 2.2 Ionization Methods………………………………………………… 9 2.2.1 Electrospray Ionization (ESI) …………………………. 10 2.2.2 Matrix Assisted Laser Desorption Ionization (MALDI).. 12 2.3 Mass Analyzers……………………………………………………... 14 2.3.1 Time-of-Flight Mass Analyzer…………………………. 15 2.3.2 Quadrupole Mass Analyzer…………………………….. 19 2.3.3 Quadrupole Ion Trap Mass Analyzer (QIT)……………. 22 2.3.4 Quadrupole Time-of-Flight (Q/ToF) Mass Analyzer…... 25 2.4 Detectors………………………………………………………….… 27 viii 2.5 Ion Mobility Mass Spectrometry (IMMS)………………………….. 28 2.6 Tandem Mass Spectrometry……………………………………….... 34 III. MATERIALS AND INSTRUMENTATION……………………………… 37 3.1 Materials……………………………………………………………. 37 3.2 Instrumentation…………………………………………………….. 39 3.2.1 Esquire-LC ESI-QIT…………………………………… 39 3.2.2 Synapt HDMS Ion Mobility Mass Spectrometer………. 41 3.2.3 MALDI-Q/ToF Mass Spectrometer……………………. 42 3.2.4 Ultraflex III ToF/ToF Mass Spectrometer……………… 43 IV. MASS SPECTROMETRY CHARACTERIZATION OF POLY(ETHYLENE GLYCOL)S SYNTHESIZED BY GREEN CHEMISTRY……………………………………………………………… 45 4.1 Background………………………………………………………… 45 4.2 Sample Preparation and Instruments Used………………………… 48 4.3 Characterization of PEG/TEG Biomaterials……………………….. 49 4.3.1 Characterization of PEG/TEG Diacrylate by ESI-QIT and MALDI ToF/ToF MS……………………………… 50 4.3.2 Characterization of Tetrahydroxy Substituted Dendritic TEG by ESI QIT…………………………….. 59 4.3.3 Characterization of TEG Dimethacrylate by ESI-QIT MS…………………………………………… 64 4.3.4 Characterization of mPEG-VA by MALDI-ToF/ToF MS…………………………………. 66 4.4 Conclusion…………………………………………………..……... 73 ix V. INVESTIGATION OF THE ENZYMATIC DEGRADATION OF POLY(LACTIDE) BY MASS SPECTROMETRY METHODS………………………………………………………………... 75 5.1 Background……………………………………………………….. 75 5.2 Sample Preparation, Instrument Used and Enzymatic Degradation……………………………………………………….. 76 5.3 ESI-Q/ToF MS Characterization of the Products from the Enzymatic Degradation of PLA……………………………….. 77 5.4 Conclusion…………………………………………………………. 88 VI. PROBING ADSORPTION ON ELECTROSPUN THERMOPLASTIC ELASTOMERIC SURFACES VIA MASS SPECTROMETRY………………………………………………………... 90 6.1 Background………………………………………………………... 90 6.2 Sample Preparation………………………………………………… 93 6.3 Electrospining……………………………………………………… 94 6.4 Compression Molding……………………………………………... 94 6.5 Protein Adsorption………………………………………………… 95 6.6 Calibration Curves………………………………………………… 96 6.7 Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS)……………………. 96 6.8 Scanning Electron Microscopy (SEM) Imaging………………….. 97 6.9 Water Contact Angle (WCA) Measurement……………………… 97 6.10 Reasults and Discussion…………………………………………. 98 6.10.1 Calibration Results……………………………………. 98 6.10.2 Surface Hydrophobicity and Morphology…………….. 101 6.10.3 Protein Adsorption Behavior of D_IBS and PS Surfaces at Different pH Levels………………………. 104 x 6.11 Conclusion………………………………………………………… 110 VII. TOP-DOWN MULTI-DIMENSIONAL MASS SPECTROMETRY METHODS FOR SYNTHETIC POLYMER ANALYSIS……………….. 113 7.1 Background………………………………………………………... 113 7.2 Experimental……………………………………………………….. 115 7.2.1 Materials………………………………………………..