ACE Phenotyping in Human Blood and Tissues: Revelation of ACE Outliers and Sex Differences in ACE Sialylation

Round 1

Reviewer 1 Report

The study investigates into characterizing Angiotensin-Converting Enzyme (ACE) phenotypes in patients with cardiovascular diseases through a novel method called ACE phenotyping. It examines ACE activity, immunoreactive protein levels, and conformation in tissue and serum samples from 50 patients, aiming to identify unique ACE phenotypes and gender differences in sialylation of ACE glycosylation sites. However, potential biases or confounding factors that may influence result interpretation should be considered.

The inclusion and exclusion criteria for patient selection must be explicitly defined to ensure the study's validity.

Although the study enrolls 50 patients, it only conducts whole exome sequencing from 8 patients. The rationale for selecting this subset from the larger sample pool should be clarified, likely relating to the focus on specific genetic variations or markers.

Differentiating from the authors' prior work, such as "Urinary ACE Phenotyping as a Research and Diagnostic Tool: Identification of Sex-Dependent ACE Immunoreactivity," this study likely extends the investigation into ACE phenotyping to tissue and serum samples, broadening the scope of understanding.

Limitations of the study, such as the small sample size for whole exome sequencing, must be acknowledged.

Regarding ACE phenotyping, there seems to be a discrepancy between the abstract and methodology sections regarding the number and types of samples subjected to analysis. This discrepancy needs clarification to ensure the accuracy of the study's methodology.

Overall, the methodology should provide clear details on the number and types of samples subjected to ACE phenotyping to avoid confusion and ensure transparency in the research process.

Minor improvement is needed

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

I would like to recommend this manuscript for publication, but several small problems should be corrected:

1. There are too many keywords in the manuscript, are each one a keyword?

2. Please unify the interval punctuation in Keywords, is it a semicolon or a comma?

3. In Section 2.8 Statistical analysis, "means ± SD from 2-5 independent experiments (depending on individual)

with triplicates", how to obtain triplicates from 2 experiments

4. A western blot or PCR characterization is suggested to prove the ACE is used.

5. Author Contribution should be declared in the end of the manuscript.

6. Due to the use of human blood samples in the article, authorization

from the ethics committee is required.

7. Is the molecular structure simulation diagram in Fig. 4-Fig.7 calculated

by the authors themselves or cited from literature? If it is cited from literature,

relevant copyright permissions are required.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have made substantial changes in several part of the paper and addressed the reviewers’ comments. This manuscript may be accepted for publication.

Author Response

Official permission from the original creators (PLOS One) of figure 6.

Link to PLOS One Policy:

https://journals.plos.org/plosone/s/licenses-and-copyright#loc-give-proper-attribution

and excerpt from their policy itself:

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We properly cited in the legend to Fig.6F:

1. F. Model of ACE dimer (adapted from [25]) where docking of HSA (from Fig.5)
2. Danilov, S.M.; Gordon, K.; Nesterovitch, A.B.; Luensdorf, H.; Chen, Z.; Castellon, M.; Popova, I.A.; Kalinin, S.;

Mendonca, E.; Petukhov, P.A.; Schwartz, D.E.; Minshall, R.D.; Sturrock, E.D.

Angiotensin I-converting enzyme mutation (Y465D) causes dramatic increase in blood ACE

via accelerated ACE shedding due to changes of ACE dimerization. PLoS One. 2011, 6, e25952.

Author Response File: Author Response.docx