Background and Aim: Developing a robust non-biopsy approach for diagnosing celiac disease (CeD) depends on identifying highly dependable, non-invasive markers of villous atrophy.
Patients and Methods: Paired duodenal mucosal biopsies were obtained from 30 patients with CeD, both untreated and following a gluten-free diet (GFD), alongside 25 disease controls. For the global identification and quantification of differentially expressed proteins (DEPs), samples were analyzed via Data-Independent Acquisition (DIA)-based SWATH-MS using a QTRAP 6500 plus mass spectrometer (AB SCIEX). Candidate DEPs were subsequently selected using the random forest-based BORUTA algorithm. Internal validation of these targets was performed via Multiple Reaction Monitoring (MRM)-based targeted proteomics. To ensure reproducibility, external validation was carried out in an independent cohort comprising 30 CeD patients (pre- and post-GFD treatment) and 46 disease controls. Finally, the plasma expression levels of the top candidate biomarkers were evaluated by ELISA in a separate validation cohort.
Results: A total of 3,424 proteins were identified in mucosal biopsies, from which 46 candidate biomarkers were shortlisted. Of these, 32 proteins were successfully optimized for MRM validation utilizing synthesized heavy- and light-isotope-labeled peptides. External cohort validation confirmed eight proteins that were significantly downregulated in untreated CeD patients compared to controls, all of which showed expression reversal following a GFD: intestinal-sucrase isomaltase, intestinal fatty acid-binding protein, intestinal-type alkaline phosphatase, mucin-13, fructose-1,6-bisphosphatase 1, mitochondrial ornithine aminotransferase, aldolase B, and meprin 1A. Six of these candidates (sucrase-isomaltase, aldolase B, fatty acid-binding protein 2, mucin-13, meprin 1A, and ornithine aminotransferase) were successfully validated via plasma ELISA. Interestingly, plasma levels of these six proteins were significantly elevated in untreated CeD patients and became significantly downregulated upon adherence to a GFD.
Conclusion: Altogether, we identified eight diagnostic proteins with the potential to monitor enteropathy non-invasively. Nonetheless, future studies utilizing larger independent cohorts are necessary to confirm their clinical utility.