title: Using mass spectrometry to detect hydrolysed gluten in beer that is responsible for false negatives by ELISA.
abstract: 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.
authors.affiliation: CSIRO Agriculture Flagship, 306 Carmody Road, St Lucia, QLD 4067, Australia. Electronic address: michelle.colgrave@csiro.au.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Colgrave
authors.affiliation: CSIRO Agriculture Flagship, 306 Carmody Road, St Lucia, QLD 4067, Australia.
authors.forename: Hareshwar
authors.initials: H
authors.lastname: Goswami
authors.affiliation: CSIRO Agriculture Flagship, GPO Box 1600, Canberra, ACT 2601, Australia.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Blundell
authors.affiliation: CSIRO Agriculture Flagship, GPO Box 1600, Canberra, ACT 2601, Australia.
authors.forename: Crispin A
authors.initials: CA
authors.lastname: Howitt
authors.affiliation: CSIRO Agriculture Flagship, GPO Box 1600, Canberra, ACT 2601, Australia.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Tanner
mesh_terms: D000595:Amino Acid Sequence; D001515:Beer / Q000032:analysis*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005188:False Negative Reactions; D005983:Glutens / Q000032:analysis* / Q000737:chemistry; D006868:Hydrolysis; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005983:Glutens
keywords: ELISA; Gluten; Mass spectrometry (MS); Multiple reaction monitoring (MRM); Quantification