An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease

Celiac disease (CeD) is a common autoimmune disorder induced by consuming gluten proteins found in wheat, barley, and rye. Glutens resist breakdown by gastrointestinal proteases, ultimately resulting in peptides with chemical structures that elicit inflammation in patients with CeD. Despite well-established connections between digestion-resistant glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, which have impeded our knowledge of potentially diverse peptide species that encounter the immune system. Here, we developed a novel liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. Using this protocol, we detected 679 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and 5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differed from control subjects. Our urinary peptidomic workflow provides new foundations for understanding gluten immunogenicity and enhancing CeD management. Additionally, it should promote new understandings of the uncharacterized dietary and urinary peptidomes.