Integrative Proteomics and Phosphoproteomics Profiling of Chronic Enteropathy Associated with SLCO2A1 Gene Reveals Mucosal Barrier Impairment and Focal Adhesion Pathway Alterations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Materials and Methods

Why were samples collected from only three patients? CEAS is a rare condition, but there is no shortage of patients with CD or healthy mucosa.

2. The lables in Figures 4 and 5 are illegible. Please make them more legible.
3. If possible, combine the discussion section with the results section.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. The sample size is very limited (n=3 per group), which significantly reduces statistical power and generalizability. The authors should justify the cohort size and discuss its implications for biological interpretation.
2. Differential expression analysis used nominal p-values without multiple-testing correction, increasing the likelihood of false positives. The authors should provide FDR-adjusted analyses or supplementary corrected statistics.
3. The justification for excluding CD-specific proteins from downstream analysis is insufficient, as these could provide valuable insights.
4. Independent validation experiments are lacking. Key proteins like POSTN, TLN1, CDH1, and VIM should be validated through immunohistochemistry, western blotting, or qPCR.
5. The interpretation of focal adhesion dysregulation as a key pathogenic mechanism lacks demonstrated causality and requires functional assays for support.
6. The molecular docking section seems disconnected from the main study and lacks clarity regarding the selection of only four proteins for docking with PGE₂.
7. Key clinical patient information is missing, including disease duration, medication history, and severity, all of which could affect proteomic profiles.
8. Potential confounding from FFPE tissue processing is not adequately addressed and may affect results.
9. The discussion overstates the translational significance of the identified hub genes and should moderate claims about therapeutic targets.
10. Some enrichment analyses rely on small protein subsets, and the authors should discuss the robustness and validity of these findings more cautiously.
11. Phosphoproteomic GSEA results are mostly negative, yet the discussion emphasizes phosphorylation signaling abnormalities; this inconsistency should be reconciled.
12. Figure legends lack methodological details, which hinders reproducibility. Improvements in clarity are needed.
13. A clearer definition of "healthy controls" is required, along with the sources and clinical backgrounds of these tissues.
14. Raw proteomics/phosphoproteomics quality control metrics are insufficiently reported; additions should include peptide coverage and missing-value handling.
15. The manuscript contains repetitive terminology, particularly around "focal adhesion dysregulation" and "epithelial barrier impairment," which could be streamlined for clarity.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I thank the authors for the opportunity to review this interesting manuscript. The work evaluates chronic enteropathy associated with SLCO2A1 gene, a rare disease that may be difficult to distinguish from Crohn’s disease. The authors explored proteomic and phosphoproteomic profiles of intestinal mucosa, aiming to identify pathways potentially associated with CEAS pathogenesis. The study is interesting, especially by pointing to alterations related with epithelial barrier, extracellular matrix remodeling and focal adhesion pathways.  

Notwithstanding the interest of the work, some aspects should be improved.  

First, the number of samples is very reduced, with only three individuals per group. This is understandable due to the rarity of CEAS, but strongly limits the conclusions. Therefore, the manuscript should be presented as an exploratory pilot study. It would be relevant to include a short table with the clinical and sample characteristics, namely age, sex, SLCO2A1 variants, disease duration, lesion location, treatment, inflammatory status, sampling site and FFPE block information.  

Second, regarding the statistical analysis, the use of fold-change thresholds and nominal P values is a limitation, considering the high number of proteins and phosphosites analyzed. The authors should present adjusted P values/FDR values, or at least a sensitivity analysis after multiple-testing correction. If only few results remain significant, the conclusions should be more cautious.  

Third, the methodological description should be improved. It is important to clarify normalization, missing values, imputation, protein inference, quality-control data, number of proteins and phosphosites identified before and after filtering, and if batch effects were evaluated. In phosphoproteomics, it should also be clarified if phosphorylation changes were normalized to total protein abundance.  

Fourth, the biological interpretation should be moderated. POSTN, CDH1, TLN1 and VIM are interesting hub genes, but the identification as hub genes in PPI networks does not prove causality, diagnostic value or therapeutic relevance. These proteins should be presented as candidate findings, unless additional validation is performed, for example by immunohistochemistry, immunofluorescence, Western blotting or targeted proteomics.  

Fifth, the molecular docking analysis needs a better biological justification. Although supplementary results are presented, it is not completely clear why PGE₂ was docked to structural or cytoskeletal hub proteins, since PGE₂ effects are usually mediated by transporters, receptors and downstream signaling. This section should be better justified or presented only as a limited exploratory analysis.  

Finally, some figures are difficult to read, namely volcano plots, enrichment plots and PPI networks. These figures should be enlarged or simplified. Figure legends should also indicate the thresholds used and whether P values are nominal or adjusted. The supplementary material statement should be checked to match the real supplementary files.  

Overall, this is an interesting exploratory study on a rare disease. However, before publication, the manuscript needs clearer clinical characterization, stronger statistical analysis, more methodological detail and more cautious conclusions. My recommendation remains: reconsider after major revision.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have successfully addressed the issues raised and I recommend accepting the manuscript for further publication.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 Thank you for your hard work on this revision.

The authors have successfully addressed all of my previous concerns. The changes made have significantly improved the overall quality of the manuscript. In its current state, I am pleased to recommend this article for publication.