Proteomics approaches to developing mass spectrometry methods for detection of gluten in gluten-free foods

Abstract

Coeliac disease and IgE-mediated allergies to wheat affect at least 1% of the global population, and as such are important diseases. As no cure currently exists for either of these diseases, patients are advised to avoid all cereals containing gluten (wheat, barley, rye and oats). However, information on the relative potency of different cereal species to cause coeliac disease or IgE mediated allergies is lacking, as is knowledge of how processing can affect the generation of immunoreactive peptides. In order to address these gaps in knowledge, curated protein sequence databases were created of seed storage prolamins from cereals containing gluten, and the relative burden of coeliac toxic motifs was assessed. This analysis showed almost all gluten protein types carry coeliac toxic motifs, with alpha-gliadins of wheat, gamma-type prolamins in wheat and barley, and omega-type prolamins in wheat, barley and rye possessing the highest number of motifs. This in silico analysis also showed wheat carried the greatest burden of coeliac toxic motifs. Proteomic profiling was then undertaken to verify this observation. Initially, extraction of gluten proteins from wheat flour was optimised and although no one method was able to extract all proteins, a simple single-step procedure was identified as being suitable for application in proteomic analysis. The optimised extraction procedure was therefore applied in the proteomic analysis of cereals containing gluten which confirmed the in silico ranking of gluten protein types in terms of coeliac toxicity, and that wheat has much higher content of coeliac toxic motifs than oats. Allergens causing IgE reactions followed a similar pattern. Proteomic profiling allowed a suite of species-specific peptide markers for gluten to be identified which were able to confirm the very low level of gluten in oats, although it lacked the sensitivity for analysis of gluten-free products. Finally, the generation of immunoreactive peptides from a processed product was assessed through the simulated gastro-intestinal digestion of a pizza base fortified with soy paste. This revealed the generation of immunoreactive peptides in both the gastric and intestinal phases with long, immunologically relevant peptides to coeliac disease in particular persisting. The data presented show oats may pose a very small risk to those with coeliac disease and IgE mediated allergy to wheat and justify further clinical research to confirm this observation.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Chiara Nitride (Supervisor) & Elizabeth Clare Mills (Supervisor)