PONE-D-21-30987Quantitative glycoproteomics of human milk and association with atopic diseasePLOS ONE

Dear Dr. Jarvinen,

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.

Both reviewers expressed concerns in finding clear explanations on some key points such as quantification and the 2nd review stresses more concerns on precision and rigor lacking in data analysis and interpretation that are crucial steps in the study.

Please submit your revised manuscript by Dec 23 2021 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Frederique Lisacek

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?

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: No

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: Yes

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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

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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: Overall, this is a well performed study and is well described. I think that it provides a nice set of information for the field which may lead to future research examining the potential impact of the associations between site-specific glycosylation of milk proteins and allergy.

Abstract

32: N-glycopeptide should be N-glycoprotein—as you are using tryptic peptides to identify the glycoproteins.

34: same comment. Should be glycoprotein, not glycopeptide profiles. Saying glycopeptide implies that you studied the naturally occurring glycopeptides rather than the trypsin-produced peptides.

Intro:

93: you are actually comparing N-glycoprotein profiles (via tryptic N-glycopeptides), not N-glycopeptide profiles—the N-glycopeptides here are created in your study, not a natural phenomenon.

Results

Table 2: max fold change and P-value numbers shoul have decimal points as periods, not commas. Same for Table 3.

Method

424: Should give time and temperatures for centrifugation steps.

433: iodoacetamide should not be capitalized.

Statistics: It seems that a correction for multiple testing should be applied (each test increases the likelihood of a false positive). I do not see one listed.

Reviewer #2: Data interpretation: The abstract is overstretched. “These four glycopeptides may have a protective effect against the development of atopic disease. Our findings indicate that the differential glycosylation of milk proteins may affect the development of atopic disease, something previously uninvestigated.” As the authors do present a mere association, yet no mechanistic hypothesis, and also no experimental support for a causal relationship whatsoever, these final sentences of the abstract appear poorly supported. Please adjust.

Data interpretation: what are the differences between the milk samples? Please try an distill from the data some major changes: how is site-specific glycosylation different, is e.g. sialylation up or down, are there differences in fucosylation?

To which extent are the differences in glycopeptide signals caused by differences in the expression of proteins, versus differences in site-specific glycosylation? An integrated proteomic and glycoproteomic analysis of the data would be necessary.

Glycopeptide identification: The authors mention that MS/MS assignments were manually checked, but they do not display any of the MS/MS spectra. Please provide assigned MS/MS spectra as supplementary data for all de novo assignments and for all more exotic compositions and proposed structures.

Figure 1: the H3N3S1 glycan structure is not credible, with the sialic acid linked to GlcNAc; please adjust

Figure 2: what is the structural basis for assigning fucoses to the core, to the antennary GlcNAc, or antennary galactose; please provide support for these assignments, same for figure 3.

Quantification: quantifying each charge state separately does not make sense, as the aim is to quantify glycopeptides, not MS signals. Hence, please sum up the different charge states before quantification, or rely on the most intense one.

The quantification needs some clarification: For example, it is unclear how the fold change was calculated, as the tables have a column headed Max fold change. What is a Max fold change ? I searched the manuscript for explanation, but could not find it.

In line with this comment, please provide comprehensive legends for all suppl. Tables to guide the reader in assessing the data.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-30987R1Quantitative glycoproteomics of human milk and association with atopic diseasePLOS ONE

Dear Dr.Jarvinen,

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. The revised manuscript still does not include enough evidence supporting some claims in the text. This requires according to reviewer #2, the enhancement of supplementary material and the inclusion of further details justifying some structural assignments.

Please submit your revised manuscript by April 15, 2022. 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,

Frederique Lisacek

Academic Editor

PLOS ONE

Journal Requirements:

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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. 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: (No Response)

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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4. 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

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5. 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

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6. 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: All changes requested have been made accurately. The manuscript is now ready for publication in the journal.

Reviewer #2: The authors have considerably improved the manuscript by toning down on some statements, and including more information in e.g figure and table legends. A lot of the key data of the paper are, however, not sufficiently accessible, and I therefore propose a major revision of the manuscript, to make some of the conclusions more substantiated, make the paper more comprehensible, and ultimately increase its impact.

1. Display of MS/MS data. The authors indicate that MS/MS data are made online available. I think this is not sufficient. For the most "exotic" assignments the authors should provide fully assigned spectra to support their conclusions. I would e.g. love to see the assigned MS/MS spectrum of S2H3N10F5 now indicated in Figure 2. Also for the small number of additional, de novo assigned glycopeptides, a display of the assigned spectra may help to support the claims.

2. Another aspect of concern is the use of "Max fold change". I think one can safely agree that "Max fold change" is not a conventional way of assessing differences between groups, and is not the most meaningful parameter. Obviously, I am not interested in seeing the maximum difference between groups, but the differences of medians or means in these cross-sectional comparisons. Displaying such averages and ranges (e.g. interquartile ranges, or standard deviations) would make the paper biologically way more meaningful.

3. As a reader I would need more relevant information in the supplemental tables: what is the score column? Were the p-values corrected? If so, in which manner, with which factor? Which significance threshold was applied?

4. For some glycopeptides the antennary fucoses were assigned to galactoses, for other to N-acetylglucosamine. I would love to see the data that support this assignment, in the form of some exemplary MS/MS spectra. I would be particularly interested to know whether the authors saw diagnostic B ions such as fucose-hexose, that would substantiate such a claim.

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7. 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: No

Reviewer #2: No

[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.

Quantitative glycoproteomics of human milk and association with atopic disease

PONE-D-21-30987R2

Dear Dr. Jarvinen,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Frederique Lisacek

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. 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 #2: Partly

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

Reviewer #2: Yes

**********

4. 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 #2: Yes

**********

5. 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 #2: Yes

**********

6. 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 #2: (No Response)

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7. 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 #2: No