Food for thought: Selecting the right enzyme for the digestion of gluten

Gluten describes a complex mixture of proteins found in wheat, rye, barley and oats that pose a health risk to people affected by conditions such as coeliac disease and non-coeliac gluten sensitivity. Complete digestion of gluten proteins is of critical importance during quantitative analysis. To this end, chymotrypsin was investigated for its ability to efficiently and reproducibly digest specific classes of gluten in barley. Using proteomics a chymotryptic peptide marker panel was elucidated and subjected to relative quantification using LC-MRM-MS. Thorough investigation of peptide markers revealed robust and reproducible quantification with CVs <15% was possible, however a greater proportion of non-specific cleavage variants were observed relative to trypsin. The selected peptide markers were assessed to ensure their efficient liberation from their parent proteins. While trypsin remains the preferred enzyme for quantification of the avenin-like A proteins, the B-, D- and γ-hordeins, chymotrypsin was the enzyme of choice for the C-hordeins.