ResearchOnline@JCU This file is part of the following reference: Koeberl, Martina (2015) Identification and quantification of allergenic tropomyosin from shellfish. PhD thesis, James Cook University. Access to this file is available from: http://researchonline.jcu.edu.au/45960/ The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owner of any third party copyright material included in this document. If you believe that this is not the case, please contact [email protected] and quote http://researchonline.jcu.edu.au/45960/ Identification and Quantification of Allergenic Tropomyosin from Shellfish Thesis submitted by Martina Koeberl (Magistra rer. nat.) For the degree of Doctor of Philosophy College of Public Health, Medical and Veterinary Sciences James Cook University Townsville, Australia July 2015 ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my supervisor Andreas for giving me the opportunity to join James Cook University to obtain a doctoral degree. Thank you Andreas for guidance, support and help throughout the whole project as well as sharing some privacy to make everyone welcome in Townsville. Moreover, to present me such a new and interesting research project and make my project reality with the help from NMI and JCU. Secondly, I would like to thank the National Measurement Institute. Especially I would like to thank James from NMI for his personal interest in allergy research and pushing the boundaries to apply new techniques for allergen analytics and therefore organising funding and support to realise my PhD research project. Moreover, I would like to thank Saman from NMI, who always coped with a smile to the instrumental difficulties during my PhD. I also would like to thank Dean for his support, Louise for organising time on the mass spectrometers and Jenny for the support with finances. Thirdly, I would like to thank Anas, my co-supervisor, for the support and guidance during my experiments and providing his professional advice. Moreover, I would like to thank Waters Cooperation, especially Chris, for helping with the qTOF and the thousand phone calls to solve instrumental difficulties. Furthermore, I would like to thank the team of the Molecular Immunology Group at JCU: Sandip, Michael, Shanshan, Nik, Susan, Thimo, Roni and Fiona - thanks for your support, help and friendship. Last, but definitely not least, I would thank the most important people in my life - my parents, my family and my friends. These are the people who supported me all my life and made me who I am today. iii STATEMENT AND CONTRIBUTION OF OTHERS Statement and contribution of others in the thesis Nature of Contribution Name and Affiliation Assistance Project plan and A/Prof. Andreas Lopata, James Cook development University James Roberts, National Intellectual Measurement Institute support Dr. Saman Buddhadasa, National Measurement Institute Editorial Assistance A/Prof. Andreas Lopata, James Cook University Research ARC Future Fellowship (A/Prof. Andreas Lopata) Financial National Measurement Institute support NHMRC Project grant (A/Prof. Andreas Lopata) Stipend National Measurement Institute Blood sample Prof. Jennifer Rolland, Monash Data collection University collection Prof. Robyn O’Hehir, Monash University and The Alfred Hospital Recombinant protein Dr. Michael Smout, James Cook expression University Technical Mass spectrometry Dr. Anas Abdel Rahman support Chris Buck, Waters Cooperation Pat Sacchetta, Waters Cooperation iv Statement and contribution of others in the chapters Chapter 1: Introduction to mass spectrometry for allergen detection Koeberl, M.; Clarke, D.; Lopata, A. L. (2014). Next Generation of Food Allergen Quantification Using Mass Spectrometric Systems. Journal of Proteome Research. 13, (8), 3499-3509. Dean Clarke (DC), National MK and AL co-developed the aim of the review article. The Measurement Institute figures and tables were generated by MK with editorial A/Prof. Andreas Lopata (AL), James inputs from AL. Editorial inputs of the manuscript were Cook University obtained from AL and DC. Chapter 2: Molecular and immunological analysis of tropomyosin from King prawn (Melicertus latisulcatus) Koeberl, M.; Kamath, S. D.; Saptarshi, S. R.; Smout, M. J.; Rolland, J. M.; O'Hehir, R. E.; Lopata, A. L. (2014). Auto-Induction for High Yield Expression of Recombinant Novel Isoallergen Tropomyosin from King Prawn (Melicertus Latisulcatus) for Improved Diagnostics and Immunotherapeutics. Journal of Immunological Methods. 415, (0), 6-16. Dr. Sandip Kamath (SK), James Cook University MK and AL co-developed the research question. MK Dr. Shruti Saptarshi (SS), James performed the experimental work and data analysis. SK Cook University provided recombinant tropomyosin from Black Tiger prawn, Dr. Michael Smout (MS), James primers for tropomyosin and myosin light chain, the Cook University structural modelling of tropomyosins and myosin light Prof. Jennifer Rolland (JR), Monash chains. SS performed the CD spectroscopic analysis. MS University and The Alfred Hospital, provided knowledge about the auto-induction system. JR Australia and ROH provided the patient sera. The figures and tables Prof. Robyn O'Hehir (ROH), Monash were generated by MK with editorial input from AL. Editorial University and The Alfred Hospital, inputs of the manuscript were obtained from AL, SK, MS, Australia JR and ROH. A/Prof. Andreas Lopata (AL), James Cook University Chapter 3: Identifying signature peptides for allergenic tropomyosin to distinguish crustacean from molluscs Dr. Anas Abdel Rahman (ARA), King MK and AL co-developed the research question. MK Faisal Specialist Hospital and performed the experimental work and data analysis. ARA Research Center (KFSHRC) provided assistance with the selection of signature A/Prof. Andreas Lopata (AL), James peptides. The figures and tables were generated by MK Cook University with editorial input from AL. Chapter 4: Identifying possible allergen-candidates in crustacean and molluscs MK and AL co-developed the research question. MK A/Prof. Andreas Lopata (AL), James performed the experimental work and data analysis. The Cook University figures and tables were generated by MK with editorial input from AL. Chapter 5: Quantification of allergenic tropomyosin applying signature peptides Dr. Anas Abdel Rahman (ARA), King MK and AL co-developed the research question. MK Faisal Specialist Hospital and performed the experimental work and data analysis. ARA Research Center (KFSHRC) provided assistance with the validation of the LC/MRM A/Prof. Andreas Lopata (AL), James method. The figures and tables were generated by MK with Cook University editorial input from AL. Chapter 6: Analysis of food samples for allergenic tropomyosin MK and AL co-developed the research question. MK A/Prof. Andreas Lopata (AL), James performed the experimental work and data analysis. The Cook University figures and tables were generated by MK with editorial input from AL. v PUBLICATIONS ARISING FROM THESIS At the time of this thesis submission, two journal manuscripts describing the research findings from Chapters 1 and 2 were published. Koeberl, M.; Clarke, D.; Lopata, A. L. (2014). Next Generation of Food Allergen Quantification Using Mass Spectrometric Systems. Journal of Proteome Research. 13, (8), 3499-3509. (Chapter 1) Koeberl, M.; Kamath, S. D.; Saptarshi, S. R.; Smout, M. J.; Rolland, J. M.; O'Hehir, R. E.; Lopata, A. L. (2014). Auto-Induction for High Yield Expression of Recombinant Novel Isoallergen Tropomyosin from King Prawn (Melicertus Latisulcatus) for Improved Diagnostics and Immunotherapeutics. Journal of Immunological Methods. 415, (0), 6-16. (Chapter 2) vi ABSTRACT Shellfish allergy belongs to the “Big 8” food allergies and can cause anaphylaxis in allergenic individuals. In the gastronomy setting, shellfish includes two groups, namely crustacean and molluscs. Both groups include large numbers of different species, whereas all species have tropomyosin as one of the major allergens. However, the amino acid sequence of tropomyosin in different shellfish species varies greatly. The amino acid homology within crustacean is very high, whereas the amino acid homology within molluscs is much lower, followed by the amino acid homology between crustacean and molluscs. The legislation in Canada and the European Union therefore requires a different label for crustacean and molluscs on food products, to protect allergic consumers. However, currently there is no analytical method available to distinguish crustacean and molluscs allergens. Current methods for shellfish allergen detection and quantification are mainly based on antibodies utilised enzyme-linked immunosorbent assay (ELISA) techniques. All experimentally developed or commercially available ELISAs for shellfish allergen quantification are based on tropomyosin recognition. However, due to the amino acid homology between different tropomyosins, ELISAs cannot distinguish between crustacean and molluscs. Recently mass spectrometry techniques have been applied for allergen detection, allergen identification and allergen quantification to overcome the disadvantages of ELISAs. A detailed review on the current status of food allergy detection using mass spectrometry is provided in chapter 1. The main aim of this PhD thesis was to develop and validate a novel quantitative mass spectrometry method (LC/MRM) to be able to distinguish between crustacean species and mollusc species. To