Proteomic profiling of processing-induced modifications to food proteins

UoM administered thesis: Phd

  • Authors:
  • Rebekah Sayers

Abstract

Peanut allergy typically results from sensitisation to one or more integral seed storage proteins; Ara h 1, 3 or 2/6. Reactions can be triggered by as little as 3 mg protein in 10% of allergic individuals and are often severe, inducing anaphylaxis which can be fatal. Accidental exposure through unintended presence can therefore be hazardous and foods must be labelled appropriately. Thermal processing is one of the main factors affecting protein properties in food systems, including the formation of Maillard reaction products. Research has suggested a link between the allergenicity of foods and cooking methods employed. A systematic study was undertaken to assess the thermal dependence of these hazard proteins, focusing on changes to solubility and chemical modifications which may alter IgE binding. Proteomic profiling was used to assess the allergenic content of peanut products and develop alternative methods for allergen analysis to support evidence-based application of precautionary allergen labelling. Runner variety peanuts were processed (boiled, fried, roasted) and their protein content determined. Proteins extracts were characterised by 1D- and 2D-polyacrylamide gel electrophoresis, including differential in-gel electrophoresis. Proteomic profiling was undertaken using label-free analysis to assess allergen composition and investigate the formation of Maillard reaction products on the most clinically relevant proteins. Peanut allergen peptide targets were then identified and used to develop label-based quantitation methods and applied to (i) investigate effects of processing on peptide targets and (ii) determine peanut in chocolate products in comparison with a commercial ELISA kit. Orthogonal studies were performed using serum samples from peanut allergic patients obtained from the Manchester Respiratory, Allergy and Thoracic Surgery (ManARTS) Biobank. Patient IgE reactivity to peanut and processed peanut products was assessed by immunoblotting, inhibition ELISA and mediator release assays.Protein solubility was reduced by thermal processing and processed protein required harsh denaturing conditions for extraction. Qualitative analysis highlighted decreased solubility of key allergens, modifications and aggregation after heating. Proteomic profiling identified and quantified different isoforms of the major peanut allergens. The protein content of processed peanuts was reduced by boiling, specifically the 2S albumins, which transferred into the cooking water. The performance of peptides selected for targeted MRM experiments was influenced by thermal processing and the presence of cocoa phenolics. Ara h 2 peptides flanked by arginine were thermostable and may prove more reliable for quantification. Application of microfluidic separation enhanced the efficiency of target ionisation in complex matrices acquiring important sensitivity gains. Maillard modifications to clinically relevant proteins Ara h 2/6 were found within IgE binding domains in raw and processed peanuts. IgE reactivity studies confirmed reduced IgE binding capacity of extensively boiled peanut and hydrolysed protein, but this did not correlate to a reduction in mediator release in poly-sensitised patients.While effective extraction limits the efficacy of analyses, buffers used in MS analyses are more robust in analysing processed protein. Proteomic profiling provides a means of characterising and profiling of allergenic proteins including food ingredients used in clinical applications. Peptides selected for targeted analyses should be validated to assess their suitability in model foods. Cooking waters collected from extensively boiled peanuts may provide an alternative and safer immunotherapy agent for patients predominantly sensitised to Ara h 2/6.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date1 Aug 2017