Dear Dr. Warwicker,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

concerns on the modeling

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The manuscript entitled “Computational investigation suggests that the cell adhesion sub-proteome is enriched for sites of pH-dependence and charge burial, whilst some key intracellular pathways may be shielded from pH fluctuations.” Research work done by Shalaw et al., utilizes computational tools to investigate the pH predictions of computational models which detecting pH dependence and physiologically aspects of proteins in relevant human.

Exploring the characteristics of buried ionisable groups, with pK, pH charge networks might have high value predicted pH-dependence and/or charge networking of proteins, are interesting and more valuable.

--The approach taken was fascinating and excellent. In order to define the subset of enzymes and transporters (ET) and non-enzymes and transporters (NET) that contain the BCOI feature, which indicates a predicted pH-dependent function, the authors have effectively used the computational methods SASA (sacalc), beta-carotene oxygenase 1 (BCO1), and pKa (pkcalc, PROPKA3).

-However, because AlphaFold2 protein models naturally alternate between different conformations based on their oligomeric state, AlphaFold may need to be run with different settings or multiple predictions. The authors also run AlphaFold2 protomer models, which exhibit poor performance and some limitations.

-The methods were explained in a vague manner, and the corresponding results were discussed in less interactive way.

However, the paper has some merits. Beyond the potentiality of the manuscript, I have a few concerns that the authors may address.

Major:

1.Can you confirm that the CamSol approach, which determines a protein's solubility based on the physicochemical characteristics of its amino acid sequences, may be more accurate and dependable for proteins that are inherently disordered?

2. Why authors selected Semaphorins specifically not mentioned, Desmosomes calcium binding sites in

cadherin domains visualization missing, which are the molecular focus of BCOI prediction.

3. figure legend appeared in middle if the manuscript is unusual PDB statures acronyms missing.

Minor:

1. Figure legends labels need to be addressed, detailed explanation needed.

2. Can you highlight the amino acid residues on semaphorins charged networks 3-D structure figure models (Figure 9).

3. Same with the other models like Figure 9B plexins charge networks highlight the residual interactions maybe more reliable to readers.

4. 8bf4 mouse plexin B1 with VHH14 and VHH15 in figure files can apply different colors for the (VHH14 and VHH15) will be better understanding.

Consider the more relevant studies in PMID: 35247105; PMID: 35301897 are more value added to this manuscript.

Reviewer #2: This manuscript presents a comprehensive and ambitious computational study that leverages AlphaFold2 models and pKa prediction methods to analyze pH-dependence and charge burial across the human proteome. The core finding is novel, significant, and will be of broad interest to the fields of computational biology, biophysics, and cell signaling. The methodology is generally sound, the scale of the analysis is impressive, and the study provides a valuable resource for the community. However, there are several aspects require clarification and further analysis to fully support the claims and ensure the robustness of the conclusions.

1.The title of this manuscript is quite long and syntactically complex, an accurate, clear and concise title is more recommended.

2. The restriction to AlphaFold2 protomer models is a significant limitation, and is vividly illustrated by the failure to predict key pH-sensing residues located at oligomeric interfaces (e.g., in ASIC1/2, TRPV1). While the benchmark performance is good, the discussion should more forcefully address the potential for systematic false negatives in certain protein classes (e.g., ion channels, transcription factors) due to this limitation.

3.Definition and Justification of BCOI Filters: The study employs ten different filters to define a "Buried Charge of Interest" (BCOI). While this multi-faceted approach is a strength, the rationale for choosing the specific thresholds (e.g., SASA ≤ 15 Ų, ΔpKa ≥ 2, network size ≥3) needs to be more explicitly justified. A supplementary table summarizing the performance (sensitivity, specificity) of each filter against the benchmark set of 23 proteins would greatly help the reader understand the relative strengths and weaknesses of each method.

4.The GO analysis is a highlight of the paper. However, the biological interpretation of the charge networks in certain enriched categories (e.g., semaphorin domains, integrator complex) is currently speculative. The authors appropriately suggest roles in conformational buffering or mechanosensation beyond pH-sensing. This discussion should be tightened to more clearly distinguish between data-driven observation (enrichment of BCOI) and hypothesis-driven speculation (potential functional role).

5. GO analysis revealed that BCOI is enriched at the cell periphery (particularly in cell adhesion molecules, ion channels, GPCRs), while it is depleted in categories such as ribosomal and nuclear structures. These findings suggest that certain key intracellular processes may possess a "buffering" capacity against pH fluctuations. Whether these enrichment/depletion patterns are related to subcellular pH environments or functional demands?

6.In Figure 7 (GO Enrichment/Depletion)�the labels are illegible. A higher-resolution version of Figure 7 with clearly readable text is recommended. The supporting figures (S1, S2) should also be briefly described in the main text to ensure readers do not overlook them.

7.Table 2 is very helpful. Consider adding a column for the SASA value of the reported pH-sensing residue in the protomer model to help readers contextualize the prediction failures.

8.Please expand slightly on what is meant by "functional resilience" in this context.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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Computation suggests that the cell adhesion sub-proteome is enriched for sites of pH-dependence and charge burial.

PONE-D-25-42837R1

Dear Dr. Warwicker,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Xiangming Zha, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Authors carefully addressed raised concerns by reviewers now its worthy publish in Plos One Journal.

Reviewer #2: The authors have adequately addressed all my previous concerns, and the revisions have significantly improved the clarity and quality of the work. I have no further questions or comments and recommend acceptance.

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

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