Celiac disease (CD) is an inflammatory disorder of the small intestine. Gluten peptides are supposed to be responsible for the reaction, the best-researched of which is the so-called ‘33-mer’. Analogous peptides in secalins (rye) and hordeins (barley) have been described. This study presents the degradation of gliadins, glutenins, hordeins and secalins purified from the respective flours using a prolyl endopeptidase from the Basidiomycete Flammulina velutipes (FvpP). The flour fractions were incubated with the enzyme, and the cleavage sites were determined using high-resolution nLC-qTOF-MS/MS. For the wheat samples, eight cleavage sites in the 33-mer peptide were shown, and all of the six described epitopes were successfully cleaved. For the commercially available prolyl-specific endopeptidase from Aspergillus niger (An-Pep), which was used as a control, only two cleavage sites that cleaved three of the six epitopes were identified. For the secalins, four prolyl-specific cleavage sites in the CD-active peptide QPFPQPQQPIPQ were found for the FvpP but none for the An-Pep. The CD-active peptide QPFPQPEQPFPW in C-hordein was cleaved at three prolyl-specific positions by the FvpP. The study proves the usability of FvpP to degrade CD-inducing peptides in real-grain flour samples and indicates its higher effectiveness compared with An-Pep. A clinical study would be required to assess the therapeutic or preventive potential of FvpP for CD
