Malcolm Blundell

Measuring Hordein (Gluten) in Beer – A Comparison of ELISA and Mass Spectrometry

Background Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic. Methods We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS). Results Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60–140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins. Conclusions ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique, enabling the quantification of individual hordein isoforms. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.

Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference

Background Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of

Using mass spectrometry to detect hydrolysed gluten in beer that is responsible for false negatives by ELISA.

6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed. Aim The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined. Results A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation. Conclusions Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.

Proteomic Profiling of 16 Cereal Grains and the Application of Targeted Proteomics To Detect Wheat Contamination

Gluten is the collective name for a class of proteins found in wheat, rye, barley and oats. Eating gluten triggers an inappropriate auto-immune reaction in ∼70 million people globally affected by coeliac disease, where the gut reacts to gluten proteins and this triggers an immune response, resulting in intestinal inflammation and damage. Gluten-free foods are now commonplace, however, it is difficult to accurately determine the gluten content of products claiming to be gluten-free using current methodologies as the antibodies are non-specific, show cross-reactivity and have different affinities for the different classes of gluten. The measurement of gluten in processed products is further confounded by modifications to the proteins that occur during processing and in some case hydrolysis of the proteins. In this study, LC-MS/MS was used to profile whole beer, and two beer fractions representing hydrolysed hordeins (<30 kDa) and hordein peptide fragments (<10 kDa). Subsequently, multiple reaction monitoring (MRM) MS enabled the relative quantification of selected peptide fragments in beer and revealed that certain classes of hordein were prone to hydrolysis (B- and D-hordein). Furthermore, select beers contained very high levels of gluten-derived fragments. Strikingly, those beers that contained high levels of B-hordein fragments gave near zero values by ELISA. The hydrolysed fragments that persist in beer show a dose-dependent suppression of ELISA measurement of gluten despite using a hordein standard for calibration of the assay. The development of MS-based methodology for absolute quantification of gluten is required for the accurate assessment of gluten, including hydrolysed forms, in food and beverages to support the industry, legislation and to protect consumers suffering from CD.

Creation of the first ultra-low gluten barley (Hordeum vulgare L.) for coeliac and gluten-intolerant populations.

Global proteomic analysis utilizing SDS-PAGE, Western blotting and LC-MS/MS of total protein and gluten-enriched extracts derived from 16 economically important cereals was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of wheat contamination in foods. The number of proteins identified in each grain correlated with the number of entries in publicly available databases, highlighting the importance of continued advances in genome sequencing to facilitate accurate protein identification. Subsequently, candidate wheat-specific peptide markers were evaluated by multiple-reaction monitoring MS. The selected markers were unique to wheat, yet present in a wide range of wheat varieties that represent up to 80% of the bread wheat genome. The final analytical method was rapid (15 min) and robust (CV < 10%), showed linearity (R(2) > 0.98) spanning over 3 orders of magnitude, and was highly selective and sensitive with detection down to 15 mg/kg in intentionally contaminated soy flour. Furthermore, application of this technology revealed wheat contamination in commercially sourced flours, including rye, millet, oats, sorghum, buckwheat and three varieties of soy.

Identification of barley-specific peptide markers that persist in processed foods and are capable of detecting barley contamination by LC-MS/MS

Summary Coeliac disease is a well‐defined condition that is estimated to affect approximately 1% of the population worldwide. Noncoeliac gluten sensitivity is a condition that is less well defined, but is estimated to affect up to 10% of the population, and is often self‐diagnosed. At present, the only remedy for both conditions is a lifelong gluten‐free diet. A gluten‐free diet is often expensive, high in fat and low in fibre, which in themselves can lead to adverse health outcomes. Thus, there is an opportunity to use novel plant breeding strategies to develop alternative gluten‐free grains. In this work, we describe the breeding and characterization of a novel ultra‐low gluten (ULG) barley variety in which the hordein (gluten) content was reduced to below 5 ppm. This was achieved using traditional breeding strategies to combine three recessive alleles, which act independently of each other to lower the hordein content in the parental varieties. The grain of the initial variety was shrunken compared to wild‐type barleys. We implemented a breeding strategy to improve the grain size to near wild‐type levels and demonstrated that the grains can be malted and brewed successfully. The ULG barley has the potential to provide novel healthy foods and beverages for those who require a gluten‐free diet.

Comparing Multiple Reaction Monitoring and Sequential Window Acquisition of All Theoretical Mass Spectra for the Relative Quantification of Barley Gluten in Selectively Bred Barley Lines

UNLABELLED Consumers, especially those with allergies and/or intolerances, should have confidence in two critical areas of food safety: foods should be correctly labelled and free from contamination. To this end, global proteomic analysis employing LC-MS/MS of gluten-enriched extracts derived from 12 barley cultivars was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of barley contamination in foods. Subsequently, a number of candidate barley-specific peptide markers were evaluated by multiple-reaction monitoring MS. From an initial panel of 26, 9 peptide markers were unique to barley, yet present in a wide range of barley varieties. The analytical method was then used to examine a range of breakfast cereals and was able to detect barley in a barley-based breakfast cereal and a muesli, but additionally allowed detection of contamination of cereals that were comprised of ancient grains and in commercially-sourced flours, including amaranth, chia, buckwheat, millet, rice, corn, oats, rye, spelt and green wheat (0.01-0.08%). LC-MS/MS provides an alternative to ELISA approaches to monitor food safety and the identification of robust and sensitive cereal-specific peptide markers is the first step toward the adoption of this technology. SIGNIFICANCE Coeliac disease is a serious health issue affecting up to 70million people globally for which there is no cure. The only treatment is a life-long gluten-free diet. Contamination of foods can occur at many stages of food production from farm to fork. As such, accurate quantification and identification of the source (i.e. cereal) and type (e.g. gluten) of contamination is critical to the health and well-being of a subset of the population, including those affected by coeliac disease and non-coeliac gluten sensitivity.

Corrigendum to “Using mass spectrometry to detect hydrolysed gluten in beer that is responsible for false negatives by ELISA” [J. Chromatogr. A 1370 (2014) 105–114]

Celiac disease (CD) is a disease of the small intestine that occurs in genetically susceptible subjects triggered by the ingestion of cereal gluten proteins for which the only treatment is strict adherence to a life-long gluten-free diet. Barley contains four gluten protein families, and the existence of barley genotypes that do not accumulate the B-, C-, and D-hordeins paved the way for the development of an ultralow gluten phenotype. Using conventional breeding strategies, three null mutations behaving as recessive alleles were combined to create a hordein triple-null barley variety. Proteomics has become an invaluable tool for characterization and quantification of the protein complement of cereal grains. In this study multiple reaction monitoring (MRM) mass spectrometry, viewed as the gold standard for peptide quantification, was compared to the data-independent acquisition strategy known as SWATH-MS (sequential window acquisition of all theoretical mass spectra). SWATH-MS was comparable (p < 0.001) to MRM-MS for 32/33 peptides assessed across the four families of hordeins (gluten) in eight barley lines. The results of SWATH-MS analysis further confirmed the absence of the B-, C-, and D-hordeins in the triple-null barley line and showed significantly reduced levels ranging from <1% to 16% relative to wild-type (WT) cv Sloop for the minor γ-hordein class. SWATH-MS represents a valuable tool for quantitative proteomics based on its ability to generate reproducible data comparable with MRM-MS, but has the added benefits of allowing reinterrogation of data to improve analytical performance, ask new questions, and in this case perform quantification of trypsin-resistant proteins (C-hordeins) through analysis of their semi- or nontryptic fragments.

Albumin and globulin proteins of wheat flour : Immunological and N-terminal sequence characterisation

In Section 2.2 of the published version of this manuscript we stated “The successful use of this particular sandwich assay requires that he epitope (PQPQPFPQE and PQQPPFPEE) must be present twice in the analyte.” This has recently been determined to be incorrect owing to a mis-identification of the Skerritt antibody. This has no impact on the cience of the paper. The corrected text should read “The successful use of this particular sandwich assay requires that the epitope (QQGYYP) must be present wice in the analyte.” In Section 3.1 we stated “The Skerritt antibody was raised against wheat gliadin and specifically recognises the nonapeptides PQPQPFPQE nd PQQPPFPEE, whereas the Mendez R5 antibody recognises the pentapeptide sequences QQPFP, QQQFP, LQPFP and QLPFP. Typical xamples include the B1-hordein (Uniprot: P06470 and I6SJ22) peptide fragments QPQPYPQQPFPPQ and PQQPFPQQPPFG, which contain he QQPFP epitope of the R5 antibody (bold) as well as sharing a stretch of six amino acids from the Skerritt antibody epitopes (underlined). ikewise, the B3-hordein (Uniprot: I6SW30) peptide fragment QPQPYPQQPQPFPQ also shares a stretch of six amino acids with the Skerritt ntibody epitope (underlined italics).” The corrected text should read “The Skerritt antibody (mAb 40121) was raised against wheat gliadin. A sister line from the same cell usion (mAb 41201) has been shown to specifically recognise the hexapeptide QQGYYP [1], and in theory the Skerritt antibody should ecognise the same epitope, whereas the Mendez R5 antibody recognises the pentapeptide sequences QQPFP, QQQFP, LQPFP and QLPFP. ypical examples include selected d-hordein (Uniprot: I6SW34) peptide fragments and the HMW-glutenins (Uniprot: J3S7U9 and C8CHI1) eptide fragments GQGQSGYYPTSPQQP and TSPQQGQQGQQGYYPTSPQQSGQWQ that contain 5 and 6 amino acids respectively of the kerritt epitope QQGYYP. Likewise, the B1-hordein (Uniprot: P06470 and I6SJ22) peptide fragments QPQPYPQQPFPPQ and PQQPFPQQPPFG ontain the QQPFP epitope of the R5 antibody (bold).” In Section 3.2 of the Results we stated “As noted above, several of the B-hordein fragments contained stretches of amino acids with ignificant homology to the epitopes recognised by the Skerritt antibody. Specifically, one of the peptides (QQKPFPQQPPFG) monitored by RM shares a stretch of six consecutive amino acids with the Skerritt epitope (PQQPPFPEE). We hypothesised that peptide fragments such s this one could bind to the antibody suppressing the ELISA response and explaining the discrepancy noted between MS and ELISA data.” This sentence should be removed from the online published version of this manuscript.