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Detection of Food Proteins in Human Serum Using Mass Spectrometry Methods

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Detection of Food Proteins in Human Serum Using Mass Spectrometry Methods University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations, Theses, & Student Research in Food Science and Technology Food Science and Technology Department 8-2020 DETECTION OF FOOD PROTEINS IN HUMAN SERUM USING MASS SPECTROMETRY METHODS Abigail S. Burrows University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/foodscidiss Part of the Food Science Commons Burrows, Abigail S., "DETECTION OF FOOD PROTEINS IN HUMAN SERUM USING MASS SPECTROMETRY METHODS" (2020). Dissertations, Theses, & Student Research in Food Science and Technology. 109. https://digitalcommons.unl.edu/foodscidiss/109 This Article is brought to you for free and open access by the Food Science and Technology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations, Theses, & Student Research in Food Science and Technology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. DETECTION OF FOOD PROTEINS IN HUMAN SERUM USING MASS SPECTROMETRY METHODS by Abigail S. Burrows A DISSERTATION Presented to the Faculty of The Graduate College of the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Food Science and Technology Under the Supervision of Professor Philip E. Johnson Lincoln, Nebraska August, 2020 DETECTION OF FOOD PROTEINS IN HUMAN SERUM USING MASS SPECTROMETRY METHODS Abigail S. Burrows, Ph.D. University of Nebraska, 2020 Advisor: Philip E. Johnson Allergenic peanut proteins are highly resistant to digestion and are detectable by immunoassays after gastrointestinal digestion. The application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for in vivo detection of peptides originating from allergenic food proteins has not been thoroughly studied. The aim of this work was to develop an in vivo detection method for peanut proteins in serum using LC-MS/MS. The method(s) were validated by analyzing subject serum collected after peanut consumption. Three de-complexation strategies were evaluated including (1) MS acquisition settings (i.e. inclusion, exclusion lists), (2) commercial depletion kits, and (3) organic solvent fractionation by discovery LC-MS/MS. Overall, none of these approaches were successful. No improvements occurred to peanut peptide detection using inclusion and exclusion lists. The commercial depletion kits removed abundant serum proteins, however, they simultaneously depleted peanut proteins. The fractionation method was efficient in reducing sample complexity, but demonstrated variable peanut protein fractionation. Due to unsuitable de-complexing strategies, we evaluated non-depleted serum by targeted MS, including parallel reaction monitoring (PRM), multiple reaction monitoring (MRM), and MRM cubed (MRM3). We identified 10 peanut peptides, representing the major peanut allergens. The limit of detection (LOD) of the sera-peanut model matrix (10:1 (w/w)) was similar for PRM and MRM, with detection at 1.0 ppm peanut protein (4.0 ppm peanut). The MRM3 method did not provide improvements to LOD. Following development of typical targeted methods, we re-investigated PRM with increased protein loading (600 µg). Peanut peptides were detected in two subject sera (sera 1, 2) at two different time points (60, 120 minutes, respectively). However, robust method development was unsuccessful, requiring further investigations in methodology. Lastly, the intermolecular arrangements of peanut seed storage proteins were evaluated by offline size-exclusion chromatography (SEC) with discovery LC-MS/MS. Gaussian modeling was used to determine the native MW of proteins, isoforms, and complexes. The combination of Gaussian modeling and discovery LC-MS/MS of SEC fractions was a highly effective separation and identification tool. iii ACKNOWLEDGEMENTS First, I must thank Dr. Phil Johnson for his belief in my ability to pursue a Ph.D. in a new and expanding field, and providing me with abundant guidance and trust to conduct this research. Phil has provided an environment encouraging of independent research and thought, which I will continue to apply in my future research endeavors. Secondly, if not as equally important, I must thank Dr. Justin Marsh for his continual support, advice, criticisms, and friendship. I am deeply grateful for his guidance and for making me a better scientist, but not a perfect scientist. I would like to thank my committee members, Dr. Joe Baumert, Dr. Steve Taylor, Dr. Sophie Alvarez, and Dr. Vanessa Garcia Larsen for their continual guidance and imploring questions through the course of my Ph.D. education. I additionally, would like to sincerely thank Dr. Steve Taylor and Dr. Joe Baumert for the opportunity to pursue, and achieve, a Master’s degree in FARRP, which has ultimately led to the pursuance of my Ph.D., and for fostering an excellent research environment. I truly cherish my time in graduate school under your direction in FARRP. I would also like to acknowledge and thank Dr. Stef Koppelman, for his continued advice, input, and supply of subject serum samples used routinely in this research project. I must thank Julie Nordlee and Dr. Samudra Wijeratne for their friendship and support since the beginning of my graduate school career. A huge thanks to Pat Gergen for her organization and helpfulness throughout my time at FARRP. I will greatly miss stopping by your office for the occasional chat. I would also like to iv acknowledge the many other individuals in FARRP who I have interacted with throughout my many years with FARRP. I would like to thank my many lab mates throughout the years, Dr. Vera Cao, Dr. Kaye Ivens, Dr. Nathan Marsteller, Dr. Bini Ramachandran, Rachel, Shimin, Lee, Jenna, and Tengfei. An extra special thanks to Dr. Shyamali Jayasena for her many years of friendship throughout both my MS and PhD programs, for being an exceptional lab mate, for answering any lab questions I may have, and her continual book suggestions. I would also like to thank my family, Mom, Dad, Cody, Amanda, Cort, Libby, Aunt Margie, and Ila, for their unending support throughout this endeavor. I would equally like to express my sincere gratitude and thankfulness to my best bro-friends, Boone, David, Brianna, and Chip for keeping me grounded, continually making me laugh, and always allowing me to express my frustrations and excitement about life in general. Words cannot describe how grateful I am to have you four in my life. To Charity and Bubbi for calling every week with mental health check-ins throughout the course of this writing process. To Ashley, Max, and Julianne, thank you for your constant support, friendship, and unending plates of cheese and glasses of wine. To Brittany, for continuing to be my best friend since freshman year of college as a result of random roommate assignments. Who knew our lives would end up this way v and our friendship would have grown this much, but you must know you have made me a more empowered woman, advocate, and friend, of which I am truly grateful. I would also like to acknowledge the many additional friends and family members who have been with me throughout my educational career. Lastly, I must express my deepest thanks to Colton. Thank you for understanding the many late nights, stress filled weeks, and joys this past year has brought into our lives. Not many people could understand the sole dedication it takes to complete a dissertation, but you have provided me with unending comfort and love in times of stress. You have brought laughter and joy into this enduring process each and every day and I very much look forward to many more joyful days. Finally, I would like to thank FARRP for providing the funding for this project. vi TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................ x LIST OF TABLES ........................................................................................................... xiii SUPPLEMENTARY ........................................................................................................ xv CHAPTER 1: LITERATURE REVIEW ............................................................................ 1 INTRODUCTION ........................................................................................................... 1 FOOD SENSITIVITIES ................................................................................................. 3 Food Intolerances ............................................................................................. 4 Food Hypersensitivities .................................................................................... 7 PEANUT PROTEINS ................................................................................................... 16 Major Allergenic Peanut Proteins................................................................... 17 Minor Peanut Allergens .................................................................................. 25 DIGESTION, ABSORPTION, AND TRANSPORT PROCESSES OF PROTEINS .. 26 Digestion of Dietary Proteins ......................................................................... 27 Dietary Protein Absorption ............................................................................. 29 Normal Responses to Food Proteins and Oral Tolerance ............................... 36 In vivo Detection of Allergenic Food Proteins ..............................................
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