A chromatographic and immunoprofiling approach to optimising workflows for extraction of gluten proteins from flour

A - Papers appearing in refereed journals

Daly, M., Huang, X., Nitride, C., Tranquet, O., Rogers, A., Shewry, P. R., Gethings, L. A. and Mills, E. N. C. 2023. A chromatographic and immunoprofiling approach to optimising workflows for extraction of gluten proteins from flour. Journal Of Chromatography B. 1215, p. 123554. https://doi.org/10.1016/j.jchromb.2022.123554

AuthorsDaly, M., Huang, X., Nitride, C., Tranquet, O., Rogers, A., Shewry, P. R., Gethings, L. A. and Mills, E. N. C.
Abstract

Ingestion of gluten proteins from wheat, and related prolamin proteins from barley, rye, and oats, can cause adverse reactions in individuals with coeliac disease and IgE-mediated allergies. As there is currently no cure for these conditions, patients must practice avoidance of gluten-containing foods. In order to support patients in making safe food choices, foods making a “gluten-free” claim must contain no more than 20 mg/Kg of gluten. Mass spectrometry methods have the potential to provide an alternative method for confirmatory analysis of gluten that is complementary to analysis currently undertaken by immunoassay. As part of the development of such methodology the effectiveness of two different extraction procedures was investigated using wholemeal wheat flour before and after defatting with water-saturated butan-1-ol. A single step extraction with 50 % (v/v) propan-2-ol containing 2 M urea and reducing agent (buffer 1) was compared with a two-step extraction using 60 % (v/v) aqueous ethanol (buffer 2) followed by re-extraction of the pellet using buffer 1, using either wheel mixing under ambient conditions (19 ◦C) or sonication at 60 ◦C. The procedures were compared based on total protein extraction efficiency and the composition of the extracts determined using a combination of HPLC, SDSPAGE and immunoblotting with a panel of four gluten-specific monoclonal antibodies. Defatting generally had a detrimental effect on extraction efficiency and sonication at 60 ◦C only improved extraction efficiency with buffer 2. Although the single-step and two-step procedures were equally effective at extracting protein from the samples, analysis of extracts showed that the two-step method gave a more complete extraction of gluten proteins. Future studies will compare the effectiveness of these procedures when applied in the sample workflows for mass spectrometry based methods for determination of gluten in food.

KeywordsGluten; Gluten-free ; HPLC; SDS PAGE ; Immunoblotting
Year of Publication2023
JournalJournal Of Chromatography B
Journal citation1215, p. 123554
Digital Object Identifier (DOI)https://doi.org/10.1016/j.jchromb.2022.123554
Open accessPublished as ‘gold’ (paid) open access
FunderBiotechnology and Biological Sciences Research Council
Funder project or codeDFW - Designing Future Wheat - Work package 2 (WP2) - Added value and resilience
Publisher's version
Output statusPublished
Publication dates
Online10 Dec 2022
Publication process dates
Accepted26 Nov 2022
PublisherElsevier Science Bv
ISSN1570-0232

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