From the journal RSC Chemical Biology Peer review history

05-Jul-2023

Dear Dr Wong:

Manuscript ID: CB-ART-05-2023-000069
TITLE: Quantitative Mass Spectrometric Analysis of Hepatocellular Carcinoma Biomarker Alpha-Fetoprotein

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

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Reviewer 1

This work by Wong and co-workers presents a Mass Spectrometric approach for quantifying alpha-fetoprotein (AFP) with or without core fucose in complex biological samples, aiming at early diagnosis of HCC. The authors employ the interesting usage of EndoS, demonstrating a methodology with high specificity and sensitivity for protein quantification. Overall, the work is solid and exhibits sufficient novelty. However, there are several points that should be addressed before publication.

1. The authors should provide an explanation for why they selected the glycopeptide backbone VN(GlcNAc/Fucose)FTEIQK for AFP quantification. Is this peptide the sole backbone carrying the N-glycan after trypsin digestion, or did this peptide sequence outperform other glycopeptides in the MS analysis?
2. It is recommended to include additional experimental conditions considering different amounts of EndoS in the titration curve. It is possible that the curve reached saturation before 3.0 µL of EndoS was reached. Please provide clarification on this matter. Ideally, the hydrolysis mediated by EndoS at different concentrations should be performed in triplicates, and error bars should be displayed on the curve.
3. Please carefully check the figure and table numbering in the legends, as there are multiple mistakes. For example, the legend for "Fig. 4. Evaluation of EndoS performance" should refer to Fig. 2, not Fig. 4.
4. Check the reference #20 for any missing information, such as issue and page numbers.

Reviewer 2

Authors presented a method to detect one peptide(VNFTEIQK) N-Glycosylation from AFP, mimicking the AFP-L3 detection. I have one concern that if representation of one peptide for AFP-L3 enough to conclude the detection accuracy ? Can author test more peptides for AFP-L3 ?

Point-by-point Responses to the Reviewer’s Comments

Thank you for considering our manuscript for publication in RSC Chemical Biology. We appreciate the opportunity to revise the manuscript based on the valuable feedback provided by the reviewers. We have carefully evaluated their comments and suggestions and have made the necessary revisions to address their concerns. Here is a point-by-point response to each comment, in which our responses are shown in BLUE, whereas the reviewer's comments are shown in BLACK.
Reviewer 1:
Comments to the Author
This work by Wong and co-workers presents a Mass Spectrometric approach for quantifying alpha-fetoprotein (AFP) with or without core fucose in complex biological samples, aiming at early diagnosis of HCC. The authors employ the interesting usage of EndoS, demonstrating a methodology with high specificity and sensitivity for protein quantification. Overall, the work is solid and exhibits sufficient novelty. However, there are several points that should be addressed before publication.

Reviewer comment 1:
The authors should provide an explanation for why they selected the glycopeptide backbone VN(GlcNAc/Fucose)FTEIQK for AFP quantification. Is this peptide the sole backbone carrying the N-glycan after trypsin digestion, or did this peptide sequence outperform other glycopeptides in the MS analysis?
Our response:
Alpha-fetoprotein (AFP) possesses only one N-glycosylation site. Considering this crucial aspect, we carefully selected VNFTEIQK for quantification based on two major reasons:
Firstly, after trypsin digestion, VNFTEIQK does not undergo any miss cleavage, ensuring a consistent and specific target for quantification. This characteristic contributes to the accuracy and reliability of our measurements.
Secondly, the selected peptide exhibits a significantly higher abundance than other peptides identified after trypsin digestion. In fact, VNFTEIQK is approximately 100-fold more abundant than the second most abundant peptide, with a miss cleavage. This high abundance further enhances the suitability of the selected peptide as the primary backbone for this study, ensuring robust and precise measurements.
Considering both the absence of miss cleavage and the high abundance, we have selected it as the optimal candidate for this study. This decision was supported by extensive validation experiments, where we demonstrated the consistent and accurate quantification of VNFTEIQK with/without core Fucosylation.
We appreciate the reviewer's recognition of the importance of these two factors in peptide selection, and we believe that our rationale for choosing this peptide is justified based on its uniqueness and abundance.

Reviewer comment 2:
It is recommended to include additional experimental conditions considering different amounts of EndoS in the titration curve. It is possible that the curve reached saturation before 3.0 µL of EndoS was reached. Please provide clarification on this matter. Ideally, the hydrolysis mediated by EndoS at different concentrations should be performed in triplicates, and error bars should be displayed on the curve.
Our response:
We appreciate the reviewer's suggestion to perform the experiment triplicate to strengthen the statistical significance of our results. We understand the importance of replicating experiments to ensure robustness and reliability. However, based on our previous experiments and publications, any volume lower than 3 μL was found to be insufficient for achieving complete removal of the outer glycan. Therefore, the resulting data obtained with lower volumes were not suitable for accurate quantification, and we did not extensively analyze those datasets. In order to ensure the completeness of the outer glycan removal, we applied 3 μL of EndoS for the rest of the experiments presented in this manuscript without further change. This consistent usage of 3 μL throughout the manuscript further supports the robustness and reproducibility of our method.
While we are unable to provide additional data at this point, we believe that our study highlights the critical role of 3 μL in achieving consistent and reliable results for the removal of the outer glycan. We acknowledge the limitations in terms of providing further data for the current manuscript. However, we recommend the use of 3 μL in future studies to ensure the reproducibility and robustness of the method.

Reviewer comment 3:
Please carefully check the figure and table numbering in the legends, as there are multiple mistakes. For example, the legend for "Fig. 4. Evaluation of EndoS performance" should refer to Fig. 2, not Fig. 4.
Our response:
We have thoroughly reviewed the legends and made the necessary corrections to ensure accurate referencing of the figures and tables. Specifically, we have carefully checked and revised the numbering in the legends to align with the actual figures and tables in the manuscript.
The legend for "Fig. 4" has been corrected to refer to "Fig. 2," addressing the reviewer's specific example. We have diligently cross-checked all other figure and table references in the legends to rectify any additional mistakes. These corrections have been incorporated in the revised manuscript to ensure the clarity and coherence of our work.

Reviewer comment 4:
Check the reference #20 for any missing information, such as issue and page numbers.
Our response:
Thank you for pointing out the missing information in reference #20. We have revisited the reference and have now obtained the missing information, including the issue and page numbers. The reference has been updated accordingly in the revised manuscript, ensuring the accuracy and completeness of our citation.

Reviewer 2:
Comments to the Author
Authors presented a method to detect one peptide(VNFTEIQK) N-Glycosylation from AFP, mimicking the AFP-L3 detection. I have one concern that if representation of one peptide for AFP-L3 enough to conclude the detection accuracy ? Can author test more peptides for AFP-L3 ?
Our response:
We appreciate the reviewer's comment and would like to clarify the objective and scope of our study. Our aim was to develop an alternative method for accurate detection of AFP with core fucosylation.
Furthermore, the accuracy and precision of our method have been thoroughly evaluated according to the guidelines set by the US FDA. Our methodology adheres to the rigorous standards outlined by these guidelines, ensuring the reliability and robustness of our results.
We appreciate the reviewer's insight regarding the suggestion of using the MS method to detect other peptides for protein quantification. While our current manuscript does not encompass the quantification of other peptides, we recognize the potential value of such an approach and will consider it for future studies.

We have carefully incorporated the suggestions and revisions as outlined by the reviewers. In the revised manuscript, we have highlighted the changes made to facilitate easy identification of the modifications. We believe these revisions have significantly improved the quality and clarity of our work.

22-Aug-2023

Dear Dr Wong:

Manuscript ID: CB-ART-05-2023-000069.R1
TITLE: Quantitative Mass Spectrometric Analysis of Hepatocellular Carcinoma Biomarker Alpha-Fetoprotein

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 2

Author responded to my question.