Dear Dr. Hosseini Ashtiani,

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Section Editor

PLOS ONE

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Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Partly

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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: No

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Reviewer #1: The article is original and very relevant for the field. This is a comprehensive metabolomics approach which analyzed OLINK proteomics data from a large epilepsy cohort in which they have previously found four differentially expressed proteins (CDH15, PAEP, LTBP3, PHOSPHO1) in patients with recent seizures. Using two machine-learning techniques, they confirmed the four previously detected proteins, but additionally uncovered ten consensus candidate protein biomarkers (CDH15, PAEP, LTBP3, PHOSPHO1, NEFL, SFRP1, TDGF1, DUSP3, WWP2 and DSG3) that contributed to classification of patients as being seizure-free or not.

The methodology of the study is modern and very complex, using very modern tools for biochemistry analysis and molecular biology.

The results conducted to the new findings showing that: using a combination of the four differentially expressed proteins (CDH15, PAEP, LTBP3, PHOSPHO1) as well as the additional consensus proteins (SFRP1, DSG3, DUSP3 and WWP2) not present among their previous findings out performed individual proteins in identifying individuals with recent seizures, highlighting the potential of multi-protein profiles.

The conclusions are consistent with the evidence and arguments presented.

The references are appropriate, including some very relevant authors experience in the field. I recommend some corrections.

1. Number of lines would be very usefull for small pointed corrections.

2. Both Introduction and Discussions should be more detailed to include the other biomarkers for epilepsy.

For Introduction you may see also M. Musteata et al., 2013, The 1H NMR Profile of Healthy Dog Cerebrospinal Fluid, PLoS ONE 8(12): e81192. doi:10.1371/journal.pone.0081192 M. Armaşu, R.M.A. Packer, S. Cook, G. Solcan, H.A. Volk, An exploratory study using a statistical approach as a platform for clinical reasoning in canine epilepsy, The Veterinary journal, 2014, 202, 292-296, DOI: 10.1016/j.tvjl.2014.08.008

Reviewer #2: This study used machine learning (Random Forest and sPLS-DA) to analyze plasma proteomics data from individuals with epilepsy, aiming to identify biomarkers linked to recent seizures. Ten candidate proteins were identified, including four previously known and six novel markers. The authors suggest that combination of proteins improved seizure classification accuracy, highlighting the potential for blood-based biomarkers in epilepsy monitoring. The study is relevant as there is an unmet need for biomarkers however there are certain aspects of the work that need improvement to increase the strength of the manuscript and comprehension of the work for the reader.

1. Review manuscript for grammatical or typographical errors. Key sentences in the manuscript need to be reviewed to be written in a clearer and more concise way.

-The title of the manuscript has typos.

-This sentence has grammar errors -” Using the four candidate markers together corroborate the utility of proteomic profiling in epilepsy research and highlight the importance of studying profiles of marker expression in addition to individual proteins. “

- Page 9, last sentence has a typo.

- This sentence has grammar errors - “Some patients with epilepsy refractory to traditional epilepsy medications respond to immunotherapy, suggesting that a better understanding of immune pathways on an individual level are likely to be integral to epilepsy care in the future.”

-Key sentence to be written clearly –“ Four proteins (PAEP, CDH15, LTBP3 and PHOSPHO1) were demonstrated to have significant association with the seizure status (p-value < 0.05) using both sPLS-DA and Random Forest models besides the rest of the proteins used for classification in both classifiers that were not among our findings in the previous study (SFRP1, DSG3, DUSP3 and WWP2).”

2. Stronger reasoning is necessary for the selection of the 4 novel proteins (SFRP1, DUSP3, WWP2 and DSG3 ) since they were not significantly differentially expressed in any of the methods - sPLS-DA and Random Forest.

3. In the discussion the authors bring up “FCAR” related to inflammation, but it has not been mentioned or discussed in the data before.

- “FCAR (Fc Alpha Receptor) Binds to the Fc region of immunoglobulins alpha and mediates several functions including cytokine, superoxide radicals and inflammatory radicals.” production.

4. More information is needed in the figure descriptions and tables to include statistical tests performed for the analysis. Additionally, the AUC in the description of Figure 2 do not match with the AUC in the figure. Label correctly the figures when attaching.

5. In the discussion of Figure 3, the authors mention that the combination of four proteins outperformed any of these previously identified proteins when used individually to predict individuals with recent seizures. However, in Figure 3B, DUSP3 has an AUC=1 suggesting that the protein perfectly distinguishes between seizure and seizure-free individuals without any error. This is either a typo or an issue in the analysis.

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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.

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Reviewer #1: Yes:  G. Solcan

Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

<p>Identification of plasma proteins associated with seizures in epilepsy: A consensus machine learning approach

PONE-D-24-55733R1

Dear Dr. Hosseini Ashtiani,

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,

Stephen D. Ginsberg, Ph.D.

Section Editor

PLOS ONE

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: The authors have made substantial Improvements of the manuscript, including all the corrections suggested.

Reviewer #2: Thank you to the authors for reviewing the manuscript and addressing the comments that were made. The manuscript looks better now.

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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