Gluten proteins found in wheat are known to have a certain resistance to gastrointestinal digestion. To address the issue of accidental gluten ingestion in individuals with gluten sensitivity, it is crucial to understand how gluten peptides behave in biological samples such as gastrointestinal juices, blood plasma, or urine. However, current immunoassays used for gluten detection in food have limitations when applied to complex biological samples like duodenal fluids. In this study, our objective was to develop a mass spectrometry (MS)-based method for accurately measuring gluten peptides in human duodenal fluids.

After conducting a literature review and characterizing a gluten-containing product, we selected seven gluten peptides, including the well-documented 33-mer gluten peptide. Isotopically labeled peptides were used as references, and we designed a targeted liquid chromatography (LC) MS assay utilizing high-resolution parallel reaction monitoring (PRM). Despite extensive optimization of the LC-PRM-MS method, the low levels of endogenous gluten peptides in human duodenal fluid samples made direct detection challenging. Therefore, we incorporated an initial immunoprecipitation (IP) step. We tested several antibodies and identified one that reliably enriched the 33-mer gluten peptide, along with a few additional gluten peptides. We assessed the performance of the immuno-LC-PRM-MS assay, focusing on quantification accuracy and precision.

Our MS-based method successfully measures the 33-mer gluten peptide in human duodenal fluids, overcoming the limitations of current classical immunoassays. By combining IP and LC-PRM-MS analysis, and utilizing isotopic dilution, our approach provides sensitive, quantitative, and reproducible measurements.