Identification and quantification of allergenic tropomyosin from shellfish

Koeberl, Martina (2015) Identification and quantification of allergenic tropomyosin from shellfish. PhD thesis, James Cook University.

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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 develop the LC/MRM based method for the quantification of tropomyosin from crustacean or molluscs, a positive tropomyosin control was generated in chapter 2, namely recombinant tropomyosin from King prawn (Melicertus latisulcatus). King prawn is a commonly consumed prawn species in Australia and the amino acid sequence of tropomyosin was analysed for the first time in this thesis. The investigated tropomyosin from King prawn was registered as the novel allergen Mel l 1 and compared to the well investigated prawn species, Black Tiger prawn (Penaeus monodon). The results demonstrated that the two tropomyosins had different amino acid sequences, resulting in different IgE binding of allergenic patient's sera. The expressed recombinant tropomyosin was in the following chapters used as positive control for the development of the LC/MRM method.

To develop the LC/MRM method for the quantification of tropomyosin from crustacean or molluscs in chapter 3 and chapter 4, twenty-two commonly consumed shellfish species in Australia have been analysed by mass spectrometry (LC/qTOF). Overall, 32 different proteins were identified, when analysing raw and whole heated shellfish extracts, whereas 16 identified proteins have been previously reported as being allergenic. The main protein identified in raw crustacean was arginine kinase, whereas in whole heated crustacean it was tropomyosin. In contrast to mollusc species where three major proteins were identified in raw and whole heated molluscs extracts, namely actin, arginine kinase and tropomyosin.

Fourteen tryptic peptides derive from tropomyosin have been identified analysing extracts of 22 different shellfish species by LC/qTOF. These 14 tryptic peptide were in silico analysed using 106 different tropomyosins to identified signature peptides to distinguish crustacean and molluscs. Based on the in silico data four peptides were selected to distinguish shellfish subgroups based on tropomyosin. In detail, peptide 1 is unique for crustacean and peptide 3 is unique for molluscs. Peptide 2 is present in crustacean and cephalopods, but not in bivalves and peptide 4 is present in prawns and lobster, but not in krill and crabs, whereas the latter includes exceptions.

To develop the LC/MRM method for the quantification of tropomyosin from crustacean or molluscs in chapter 5 the four identified peptides were chemically synthesised. Applying these four chemical synthesised peptides, the LC/MRM method was successfully developed and validated for different shellfish species. As predicted in silico, the four peptides were quantified in 22 shellfish species utilising raw and whole heated extracts, with the exception of four species. Overall, the concentration of tropomyosin is higher in whole heated extracts compared to raw extracts. Moreover, in whole heated crustacean the concentration of tropomyosin is higher compared to whole heated molluscs.

The validated LC/MRM method was applied in chapter 6 for the quantification of allergenic tropomyosin in highly processed food samples. Moreover, the LC/MRM method was compared with two commercial available ELISA kits, to confirm that both chemical and antibody based methods can quantify highly processed tropomyosin. Overall, both methods can detect allergenic TM, whereas the ELISAs had difficulties quantifying mollusc tropomyosin. Moreover, the ELISAs can certainly not distinguish crustacean from mollusc in food samples. The concentrations quantified for the food samples varied for the LC/MRM method compared to the two ELISA kits, whereas the results of one ELISA kit were similar to the concentrations quantified by LC/MRM. Thus one can assume the other ELISA kit overestimates the concentration of allergenic tropomyosin in food samples.

Overall in this PhD thesis a novel quantitative LC/MRM method was developed and validated to distinguish allergenic tropomyosin from crustacean species and from mollusc species. The validated LC/MRM method was successfully applied for the quantification of allergenic crustacean tropomyosin and allergenic mollusc tropomyosin in 22 different shellfish species and for thirteen highly processed food samples. Therefore it was demonstrated that the quantification of tropomyosin by LC/MRM is a suitable, specific and sensitive alternative to currently existing antibody based methods, such as ELISAs. The work presented in this thesis provides an important contribution towards the detection and quantification of allergenic tropomyosin from crustacean and molluscs, to fulfil the international legislation requirements for processed food.

Item ID: 45960
Item Type: Thesis (PhD)
Keywords: allergens, allergies, allergy diagnostics, amino acids, auto-induction, crustacean, enzyme-linked immunosorbent assay (ELISA), escherichia coil strains (K and B), food allergies, king prawn, mass spectrometry, molluscs, MRM, peanut, proteins, quantification, recombinant allergen, shellfish, signature peptide, soy, tropomyosin, wheat
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Publications arising from this thesis are available from the Related URLs field. The publications are:

Chapter 2: Koeberl, Martina, Kamath, Sandip D., Saptarshi, Shruti R., Smout, Michael J., Rolland, Jennifer M., O'Hehir, Robyn E., and Lopata, Andreas 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. pp. 6-16.

Koeberl, Martina, Clarke, Dean, and Lopata, Andreas L. (2014) Next generation of food allergen quantification using mass spectrometric systems. Journal of Proteome Research, 13 (8). pp. 3499-3509.

Date Deposited: 05 Oct 2016 04:16
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1107 Immunology > 110701 Allergy @ 50%
11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics) @ 50%
SEO Codes: 92 HEALTH > 9204 Public Health (excl. Specific Population Health) > 920406 Food Safety @ 33%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 34%
92 HEALTH > 9202 Health and Support Services > 920203 Diagnostic Methods @ 33%
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