From Peptide to Protein: Development of Conversion Factors for the Quantification of Gluten Using Targeted Mass Spectrometry

Immuno- and mass spectrometry (MS) test methods have 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 was prepared and characterized regarding immunoglobulin E (IgE)-reactivity and protein profile, which were then used to screen a panel of gluten peptides to identify reporters suitable for use in an MS test method for gluten determination. Four peptide markers were selected and synthesized as heavy isotopically labeled versions for further evaluation. Two were derived from α-gliadin (RPQQPYPQPQPQY and QPFPQPQLPY [spanning a celiac toxic motif]), one each from γ-gliadin (GIIQPQQPAQL [spanning a celiac 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 an 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 10 mg gluten/kg food product level, showing that the prototype method and approaches described have the potential to deliver a complementary method for determination of gluten in food.