PONE-D-22-11303Species-specific identification of donkey-hide gelatin and its adulterants using marker peptidesPLOS ONE

Dear Dr. Zhang,

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.

Comments: Please carefully revise the language in the text, paying particular attention to the format of the references.

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We look forward to receiving your revised manuscript.

Kind regards,

Chun-Hua Wang

Academic Editor

PLOS ONE

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2. Thank you for stating the following financial disclosure: 

"This work was supported by Guangdong Natural Science Foundation (No. 2020A151501577, Ying Zhang) and the Open Fund of Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research (No. 2016B 030301004, Hui Cao). YZ and HC conceived and designed the study."

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4. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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. 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: Yes

Reviewer #3: Yes

Reviewer #4: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: Yes

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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

Reviewer #3: Yes

Reviewer #4: No

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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

Reviewer #3: Yes

Reviewer #4: No

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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: 1.Only Standard medicine samples DHG, CHG and PHG were purchased from from the National Institutes for Food and Drug Control (NIFDC; Beijing, China), but HHG was not purchased. Please explain the reasons.

2.Can the doping amount in donkey-hide gelatin be identified according to the existing marker peptides content determination results?

3.What is the purity of the synthetic marker peptide standard?

4.Is there really enough horse hide for adulteration in donkey-hide gelatin production?Is it of practical significance to identify the adulterated horse hide in donkey-hide gelatin?

Reviewer #2: Donkey-hide gelatin is an important traditional Chinese medicine made from donkey skin. Identifying the animal materials (donkeys, horses, cattle and pigs) in donkey-hide gelatin is challenging. The goal of this study by Zhang J. et al. is to utilize liquid chromatography–tandem mass spectrometry method to identify species-specific marker peptides, and these marker peptides can be used to discriminate the ingredient from (donkeys, horses, cattle and pigs). This reviewer realizes that the results were so interesting because the marker peptides and LC–MS/MS method showed a good specificity, sensitivity and reproducibility. This method is valuable in controlling the quality of donkey-hide gelatin. My comments to co-authors are:

1. Figure 1 and the words that describe Figure 1 from theoretical marker peptide prediction section in Materials and Methods should be in Result section, and the Figure 1a should be Figure 1A.

2. In the “Evaluation of applicability of the LC–MS/MS method” section and the corresponding result section, the authors should organize the word and data to highlight the specificity, repeatability and sensitivity of the method.

3. In the legend of Figure 2, the authors should describe the Figure in details, such as, what are the meanings of b and y? different colors? X axis and Y axis.

4. In line 303, R2 should be R2.

5. In line 366 and 367, “Unlike our study, a previous study showed certain different theoretical marker peptides.” The authors should provide references for the study.

6. In line 379 and 380, “Previous studies conducted by other researchers on the components in gelatins may explain this result.” The authors should provide references for these studies.

7. In the manuscript, “in this study” should be changed as “in our study”.

8. In the manuscript, S1 Figure should be Figure S1 and S1 Table should be Table S1.

Reviewer #3: This is a well-written paper containing interesting results which merit publication. The paper presents an LC-MS/MS method for peptide marker detection, which was applicable and demonstrated good specificity and sensitivity for the identification of gelatins. It is a topic of interest to the researchers in the related areas but the manuscript needs a bit improvement (minor revision) before acceptance for publication. My detailed comments are as follows:

1. Please note that the English letters marked on the chart in the article are consistent with those marked on the chart itself, which can be in lowercase or uppercase.

2. Fig4 can also be attached to the end of the article like S3 figure.

3. The consistency between the information displayed by S3 figure and the content described in the discussion part of the article needs to be improved. （MAI exists in samples（b）、（d），instead（b）、（c）.

4. If the marker peptide was added into HHG, the proposed LC-MS/MS method can not identify the HHG. Please try to discuss this point deeply and try to draw a possible method to assistant to identify the it.

Reviewer #4: 1． There have been some research papers on identification of donkey-hide glue and its adulterants, so this paper should show the novelty of the work in the Introduction section, compared with the literature that had been reported. I do not think it is correct that the authors stated this manuscript as “one of the first investigations to focus specifically on the identification of gelatins using marker peptides”. Please add the literatures of related studies in the introduction part.

2． Dithiothreitol often was used to destroy the disulfide bond, does the collagen contain disulfide bond? Please make sure the necessity of Dithiothreitol must be used to destroy the disulfide bond?

3． From the manuscripts, 12 marker peptides for the four gelatins were verified only by proteomic experiments, it advised that the 12 marker peptides should be validated by different sources samples.

4． It is recommended that the manuscripts be professionally edited for English language before submitting your revised manuscript version.

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Reviewer #1: Yes: Zou Xiaoxing

Reviewer #2: No

Reviewer #3: Yes: Yuming Dong

Reviewer #4: Yes: WEI Feng

[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.]

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PONE-D-22-11303R1Species-specific identification of donkey-hide gelatin and its adulterants using marker peptidesPLOS ONE

Dear Dr. Zhang,

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 Aug 29 2022 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,

Chun-Hua Wang

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.

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

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

Reviewer #3: (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 #2: Yes

Reviewer #3: (No Response)

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

Reviewer #2: Yes

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: (No Response)

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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 #2: The authors have addressed my comments and concerns. I recommend that editors accept the submission.

Reviewer #3: The authors have answered the questions well.However, there are still some other questions to be considered.

1. If this LC-MS/MS of marker peptide is not the first investigation on DHG and its adulterants(The study presented in this thesis is a comprehensive one of the first investigations to focus specifically on the identification of gelatins using marker peptides.),what is the innovation of this LC-MS/MS?Please compare the proposed method and the published LC-MS/MS method of marker peptide on the identification of DHG.

2. Please add the method of identification of DHG of Chinsed Pharmaceopoeia 2020 version and discuss the limitations of the CHP2020 method briefly and show the proposed LC-MS/MS can be helpful to improve the CHP 2020 method in Introducation part but not in the discussion part.

3. Line113-115, … Chinese Pharmacopoeia 114 and the quality of every gelatin sample complied with Chinese Pharmacopeia 115 requirements .Please add the version of the Chinese Pharmacopoeia .for example,2020 version.

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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

Reviewer #3: Yes: OK

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[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.

Species-specific identification of donkey-hide gelatin and its adulterants using marker peptides

PONE-D-22-11303R2

Dear Dr. Zhang,

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.

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Kind regards,

Chun-Hua Wang

Academic Editor

PLOS ONE