PONE-D-24-54784Exploratory Study of Serum Protein Biomarkers for Sudden Cardiac Arrest Using Protein Extension Assay: A Case-Control StudyPLOS ONE

Dear Dr. Park,

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.

Please submit your revised manuscript by Feb 28 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Nanako Kawaguchi

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

3. Thank you for stating the following financial disclosure:  [This study was supported by the Research Program funded by the Korea Disease Control and Prevention Agency (Grant No: 2017NE3300600, 2017E3300601, 2019P330800, 2021-12-202, 2022-12-204).].  Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

4. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure.

5. Please include a caption for figure 2.

6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thank you for the privilege to review this manuscript. The manuscript is well-written and insightful. I do have a few comments which can be found below:

1.I agree with the authors that as this study is retrospective in nature, causal relationships and only associations can be investigated. It is important that they have included this in their limitation.

2.AXL and TIMP-4 are known to be affected in patients with florid cardiac history (eg chronic heart failure). An increased number of sudden cardiac arrest (SCA) cases tend to be observed among these patients. Accordingly, does this confound the findings and observations made in this study?

3.In the limitations, the authors have rightfully included that blood sampling was only performed after SCA occurred, which means that the samples could be influenced by post-cardiac arrest changes. Apart from the interventions that the authors have mentioned in their manuscript, what other interventions can the authors further suggest to improve on this? This is quite a big limitation and I would like the authors to expound more on this.

Reviewer #2: The authors used well-controlled serum samples, defined as those processed within two hours of collection, to minimize the effects of delayed sample processing. Additionally, age- and sex-matched controls were employed to reduce variability from these factors. Furthermore, the authors implemented additional criteria to narrow down candidate protein biomarkers for predicting SCA. Using blood samples from 42 SCA patients and 42 matched controls, they evaluated the serum levels of 246 proteins, identifying AXL and TIMP-4 as potential biomarkers for SCA. Multivariable analysis demonstrated that both proteins could enhance predictive power when combined with traditional risk factors. The authors proposed that these biomarkers, which are involved in cardiac remodeling and extracellular matrix processes, may contribute to the early detection and risk assessment of SCA. However, as authors discussed in the paper, the study has limitations, including small sample size, restriction of quantifiable proteins.

Here are several points the author should address to provide a clearer understanding of the results and make more reliable conclusions.

1. (Page 9, Lines 182-184)

a. Among the patients’ samples, how many samples were obtained from each center? Please include detailed center information either in the main text or in a table.

b. Do the final two proteins (AXL and TIMP-4) show consistent levels across samples from each center? Please address any potential center-specific variation in your analysis.

2. (Page 10, Lines 203-205) Could you address whether delayed blood sample collection affects the levels of the proteins?

3. (Page 11, Lines 216-220) Since the authors used the PEA assay, the following points require clarification:

a. Were the case and control samples randomized before being analyzed using the assay?

b. Please provide more details on how protein levels were normalized. Was it simply log2 scaling? For instance, if we have the NPX values of Protein A and Protein B, can these different protein levels be directly compared using NPX?

c. Were replicate results obtained within the same assay panel to address analytical variation? Additionally, if the number of samples exceeded the 96-plex limit and were processed using separate kits, what normalization strategy was employed to ensure consistency across the kits?

d. The three panels included 18 overlapping proteins. How were these overlapping proteins managed? Could you present how their quantification differed across the panels? If differences were identified, how were these proteins quantified or reconciled using the differing values from the two panels?

4. (Page 12, Lines 232-242) Please include the correlation values for each protein at each extraction step in Supplementary Table 2.

5. (Page 14, Lines 291-292) Are there any sex differences in the levels of the two proteins (AXL and TIMP-4)? Please describe these differences in detail.

6. (Page 14, Lines 293-295) Please provide a detailed explanation of the color scheme used in the heatmap.

a. The color appears to be based on NPX without any normalization. Is there a specific reason for using NPX instead of Z-scores for the heatmap? Using Z-scores for each protein might provide clearer sample clustering.

b. If the color indicates NPX, the two candidate proteins (AXL and TIMP-4) seem to show a positive correlation. Please elaborate on this relationship and provide further discussion regarding its significance.

c. Could you clarify the meaning of the following statement: “Twenty-five of SCA group (59.5%) and 22 of control group (52.3%) were affiliated with the same cluster (Figure 2).” Additional discussion is needed to explain and interpret this finding.

7. (Page 14, Lines 297-299) Since BNP levels are a well-known risk factor for cardiac arrest, how does the BNP’s predictive power differ in case and control?

a. Please address prediction power of these models, if possible.

i. BNP only,

ii. 6 traditional baseline model + BNP

b. Please discuss the predication power of these models, if possible.

i. BNP + AXL

ii. BNP + TIMP-4

iii. BNP + AXL + TIMP-4

iv. 6 traditional baseline model + BNP + AXL

v. 6 traditional baseline model + BNP + TIMP-4

vi. 6 traditional baseline model + BNP + AXL + TIMP-4

8. (Page 14, Lines 300-308)

a. What is the predictive power when using only the two proteins (AXL and TIMP-4) separately or combined, without including the baseline model? Please discuss this in detail.

b. Since the authors used the entire sample to construct the models, there is a high risk of overfitting, particularly due to small sample size. I recommend splitting samples into training set and a test set (e.g., 7:3), constructing the models with the training set, and then validating their predictive power using the test set.

9. (Page 14, Lines 289-290, Page 16, Lines 339-341) For the GO analysis, inputting only two proteins reduces the likelihood of obtaining meaningful insights from common categories. As shown in Supplementary Figure 1, only one category includes both proteins (extracellular space). To draw stronger conclusions from the GO analysis, it would be beneficial to include more proteins. Therefore, I suggest conducting the GO analysis with the 97 proteins that show a strong correlation with SCA. Please provide a more detailed discussion of the results from this expanded analysis.

**********

6. PLOS authors have the option to publish the peer review history of their article (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: Yes:  Yoshio Masuda

Reviewer #2: Yes:  Jihyeon Lee

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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.

Exploratory Study of Serum Protein Biomarkers for Sudden Cardiac Arrest Using Protein Extension Assay: A Case-Control Study

PONE-D-24-54784R1

Dear Dr. Park,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nanako Kawaguchi

Academic Editor

PLOS ONE