From peptide to protein: Development of conversion factors for quantification of gluten using targeted mass spectrometry

Immuno- and mass spectrometry (MS) test methods having been used to ensure “gluten-free” food products contain less than 20 ppm of gluten. However, comparison of test method performance is difficult due to differences in reporting units. A set of wheat flour fractions were prepared and characterised regarding IgE-reactivity and protein profile which were then used to screen a panel of gluten peptides to identify reporters suitable for use in a MS test method for gluten determination. Four peptide markers were selected and synthesised as heavy isotopically labelled versions for further evaluation. Two were derived from α-gliadin (RPQQPYPQPQPQY, and QPFPQPQLPY [spanning a coeliac toxic motif]) and one each from γ-gliadin (GIIQPQQPAQL [spanning a coeliac toxic motif and IgE-epitope]) and a low molecular weight subunit of glutenin (VQQQIPVVQPSIL). Analysis of the wheat flour fractions was achieved with peptides RPQQPYPQPQPQY, GIIQPQQPAQL and VQQQIPVVQPSIL. Two methods were used to derive a set of factors for converting from peptide marker to gluten protein, one based on calculation and a second on experimental analysis using either the gliadin or glutenin protein fractions. Experimentally derived conversion factors performed better when used in a MS test method to quantify gluten in a set of wheat flour samples. Peptide VQQIPVVQPSIL showed the greatest sensitivity and when employing a glutenin fraction-based conversion factor, gave comparable results to protein levels determined using Dumas total nitrogen analysis. This peptide marker demonstrated the potential to determine gluten at a level around the 10mg gluten/kg food product level showing the prototype method and approaches described have the potential to deliver a complementary method for determination of gluten in food.