Investigating and Annotating the Human Peptidome Profile from Urine under Normal Physiological Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors offer an analysis of the human urinary peptidome. They suggest that "these findings can serve as a foundational reference for the discovery of biomarkers in various human diseases." Some additional information is suggested for them to be successful in this endeavor.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Grammatical editing of the manuscript is required. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, Elguoshy A. and Yamamoto K. et al. investigated the human peptidome profile in urine under physiological conditions using samples from 19 healthy individuals. They identified three main precursor proteins contributing to the peptidome signal: Uromodulin (7.2%), Prostaglandin-H2 D-isomerase (4.5%), and Serum albumin (3.0%).

·      The manuscript lacks clarity in several areas, which the authors should address for better comprehensibility. Additionally, it is recommended to provide the full names of the abbreviations used in the manuscript, either at the end or as a footnote, to facilitate understanding by cross-functional researchers. The abbreviations are:

CTSD (Cathepsin D)

MMP-2 (Matrix metalloproteinase-2)

PSM (Peptide Spectrum Match)

FDR (False Discovery Rate)

·      The authors employed a 5% PSM FDR cut-off, indicating medium confidence in their results. It would be beneficial to explore the effects of lowering the cut-off to 1% or 0.5% for increased confidence in their findings. Furthermore, please annotate the Excel sheet table containing supplementary results for better clarity.

·      In Figure 1, lines 202-203, the word "Protocol" is misspelled.

·      Additionally, please provide detailed information regarding the sample types, including age and gender. In Figure 3, it is noted that peptides were observed from the placenta and ovary tissue types. Specifically, out of the 19 healthy samples, how many were from pregnant females?

Comments on the Quality of English Language

The manuscript lacks clarity in several areas, which the authors should address for better comprehensibility. Additionally, it is recommended to provide the full names of the abbreviations used in the manuscript, either at the end or as a footnote, to facilitate understanding by cross-functional researchers. The abbreviations are:

CTSD (Cathepsin D)

MMP-2 (Matrix metalloproteinase-2)

PSM (Peptide Spectrum Match)

FDR (False Discovery Rate)

·      In Figure 1, lines 202-203, the word "Protocol" is misspelled.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1.     Page 3, line 114-115: Since database search without enzyme specificity is more prone to false positive hits. Is 5% PSM FDR strict enough to control the false positive hits?

2.     Page 4, line 181: Related to question 1, is the peptide identification number of 29372 severely inflated by false positive hits?

3.     Table S1-S3: Table headers are not clear enough. What do the headers “min”, “max” mean? The two “number_of” headers seem not complete.

4.     Table S2, line 34: Peptide AEDEGGEE’s retention time ranges from 3.3 to 119.2 min. Are those all true hits? There might be some false positive hits in the peptide identifications.

5.     The quantification accuracy of spectral count method is not optimal. Zhao et al. used iBAQ labeling based quantification instead in the cited reference [24]. Have you considered to validate the normal urine peptides identified in the current work by synthetic peptide standards?

6.     P12, line 397-401: please provide validation of using the five tissue-specific degraded precursor proteins identified in the urine peptidome work as biomarkers for liver and kidney disease, or cite relevant publications.

7.     Fonts are too small in Figure 5 and Figure 7.

8.     Could you please comment on the large difference of peptide profile in this work and in the other two works in Figure 6? Only a small portion of the peptides were found in common.

9.     Could you please comment on the applicability of the urine peptidome profile obtained in the current work? How can future researchers refer to the work for biomarker discovery?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have gone over the revisions and consent to the revised manuscript being published.

Comments on the Quality of English Language

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Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for including all the feedback in the manuscript. The manuscript appears to be improved with additional remarks made by the authors.

Reviewer 3 Report

Comments and Suggestions for Authors

In the current version, the authors provided thorough explanation or comprehensive revisions per reviewer's comment. I recommend to accept the manuscript in the present form.