From the journal RSC Chemical Biology Peer review history

31-Mar-2021

Dear Dr Gunning:

Manuscript ID: CB-REV-03-2021-000055
TITLE: Optical Chemosensors for the Detection of Proximally Phosphorylated Peptides and Proteins

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.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

************

Reviewer 1

In this manuscript, Gunning and a co-workers describe a comprehensive review covering optical sensing molecules/materials of proximally phosphorylated peptides/proteins. They briefly explain the biological significance of proximal phosphorylation of proteins and analytical methods used for their detection other than optical sensing. Subsequently, they clearly summarized recent progress (for two decades) in development of optical sensors useful in aqueous (physiological) conditions. I like table 1 summarizing the representatives, which is quite helpful for readers. But I feel that a few of figures are needed which show real chemical structures of phosphorylated Ser/Thr and Tyr and their peptides sequences, one of the typical signal transduction pathways involving phosphorylation/dephosphorylation, in addition to binding modes of the sensors to a proximally phosphorylated peptide/protein. These are also valuable for readers to understand the content of this review. I believe that this manuscript is expected to attract broad interest of readers in chemical biology and analytical biochemistry, and supramolecular chemistry. I support the acceptance of this manuscript after the revision.

Reviewer 2

The manuscript by Cabral et al. reviews progress over the last 10-15 years on the development of chemosensors for detection of multiply phosphorylated peptides/proteins specifically where the two phosphosites are in mutual proximity. There has been a vast amount of effort invested by the synthetic sensor community in development of receptors/sensors for phosphate, singly phosphorylated peptides/proteins and other phosphorylated bioanalytes include nucleoside mono, di and triphosphates, but far less on multiple and proximally phosphorylated peptides/proteins. The authors make a strong case that there is a need for cheap sensors in comparison to detection by MS based phosphoproteomics which is partly true e.g. where a specific (diagnostic) application is desired (please note, it I don’t think with the advent of MS instrumentation suited to single cell proteomics and ongoing improvements that the argument around sample quantity on pg 4 is sound). The authors also neglect a more rounded discussion of competitor technologies e.g. phosphorylation state-specific antibodies (see e.g. Nat Protoc 2, 2574–2581 (2007)). That said, I enjoyed reading the review – I found it informative, accessible and providing a good overview of approaches for recognition of the target phosphosites. A balanced appraisal of the various approaches, challenges and limitations with current methods within the key focus area of the review is evident and I particularly like the comparative table towards the conclusion. Despite this, I also found it frustrating having to move back and forth to visualize the structures and whilst I am not generally in favour of repetition, I wonder if showing the key structures as a figure in each of the distinct sensor classes would help? Overall, I strongly recommend publication in RSC Chem. Biol. In addition to the points above, the authors may wish to consider the following in any minor revisions.
Pg 4: Multisite phosphorylation patterns have “been” shown to be functionally significant beyond the capability of single site modification
Pg 5: Additionally, the MS/MS approach is labour intensive and requires costly instrumentation and technical expertise effectively repeats an earlier sentence.
Pg 5: This comprehensive review will cover optical sensing – I would remove “comprehensive”
Pg 9: I suggest rewording of “and hydrophobic interactions of the BODIPY core” to e.g. “and favourable hydrophobic packing of the….”; hydrophobic interactions do not exist, rather the effect that is observed arises as a result of favorable/ unfavorable non-covalent interactions between solute(s) and solvent, so is a solvophobic effect. (repeated on page 17)
In the opening section describing largely the work of Hamachi and focussing on different spacings e.g. i, i + 7 – it would be pertinent to comment on the context recognition of such a cluster presumably has secondary structure context and so there are presumably caveats with the use of peptide based models of full length proteins? (although admittedly for e.g. IDPs and aggregated peptides with regular spacing of phosphosites this is perhaps less pertinent). Alternatively this might work as well in the conclusion as an area for further exploration.

Prof Seung Bum Park
Associate Editor, RSC Chemical Biology
13th April 2021

Manuscript title: “Optical Chemosensors for the Detection of Proximally Phosphorylated Peptides and Proteins”
Author(s): Cabral, Aaron; Radu, Tudor; de Araujo, Elvin; Gunning, Patrick
Manuscript ID: CB-REV-03-2021-000055

Dear Dr. Park,

Please find attached our revised manuscript for publication. We have addressed all the reviewers’ comments in full. We thank the reviewers for their helpful suggestions and have, where necessary, modified the text. We have provided a markup copy of the manuscript with all changes.

We hope that the revised manuscript will now be acceptable for publication in RSC Chemical Biology.

Yours sincerely,

Prof. Patrick T. Gunning
Department of Chemistry & Department of Chemical and Physical Sciences
3359 Mississauga Road North
Davis Building, Rm 4046
University of Toronto
Mississauga
ON L5L 1C6
Tel: 1-647 669 2969
Email: patrick.gunning@utoronto.ca

Summary of revisions:

We have thoroughly considered and addressed the comments of referee 1.
Referee 1 - Comments to the Author
In this manuscript, Gunning and a co-workers describe a comprehensive review covering optical sensing molecules/materials of proximally phosphorylated peptides/proteins. They briefly explain the biological significance of proximal phosphorylation of proteins and analytical methods used for their detection other than optical sensing. Subsequently, they clearly summarized recent progress (for two decades) in development of optical sensors useful in aqueous (physiological) conditions. I like table 1 summarizing the representatives, which is quite helpful for readers.
Comment 1: But I feel that a few of figures are needed which show real chemical structures of phosphorylated Ser/Thr and Tyr and their peptides sequences, one of the typical signal transduction pathways involving phosphorylation/dephosphorylation, in addition to binding modes of the sensors to a proximally phosphorylated peptide/protein. These are also valuable for readers to understand the content of this review. I believe that this manuscript is expected to attract broad interest of readers in chemical biology and analytical biochemistry, and supramolecular chemistry. I support the acceptance of this manuscript after the revision.
Response to Comment 1: We thank the reviewer for their comment. We have included a figure in the introduction that shows the chemical structures of phosphorylated Ser, Thr and Tyr (Figure 1A) and an illustration of a typical phosphorylation/dephosphorylation signal transduction pathway (Figure 1B). Additionally, we have included a summary figure in the conclusion that illustrates the main binding modes of the reviewed sensors to proximally phosphorylated residues (Figure 7).

We have thoroughly considered and addressed the comments of referee 2.
Referee 2 - Comments to the Author
The manuscript by Cabral et al. reviews progress over the last 10-15 years on the development of chemosensors for detection of multiply phosphorylated peptides/proteins specifically where the two phosphosites are in mutual proximity. There has been a vast amount of effort invested by the synthetic sensor community in development of receptors/sensors for phosphate, singly phosphorylated peptides/proteins and other phosphorylated bioanalytes include nucleoside mono, di and triphosphates, but far less on multiple and proximally phosphorylated peptides/proteins.
Comment 1: The authors make a strong case that there is a need for cheap sensors in comparison to detection by MS based phosphoproteomics which is partly true e.g. where a specific (diagnostic) application is desired (please note, it I don’t think with the advent of MS instrumentation suited to single cell proteomics and ongoing improvements that the argument around sample quantity on pg 4 is sound).
Response to Comment 1: We thank the reviewer for this comment. Although it is true that MS instrumentation suited for single cell proteomics have become even more sensitive in the past few years, researchers studying the phosphoproteome still use complicated workflows and enrichment techniques to improve sample quality and access even deeper into the phosphoproteome. To address this, we have added additional information within the introduction detailing these advancements (page 4, lines 12-16).
Comment 2: The authors also neglect a more rounded discussion of competitor technologies e.g. phosphorylation state-specific antibodies (see e.g. Nat Protoc 2, 2574–2581 (2007)).
Response to Comment 2: We thank the reviewer for this comment. We have included information in the introduction section (page 4, lines 16-23) regarding phosphorylation state-specific antibodies.

Comment 3: That said, I enjoyed reading the review – I found it informative, accessible and providing a good overview of approaches for recognition of the target phosphosites. A balanced appraisal of the various approaches, challenges and limitations with current methods within the key focus area of the review is evident and I particularly like the comparative table towards the conclusion.
Despite this, I also found it frustrating having to move back and forth to visualize the structures and whilst I am not generally in favour of repetition, I wonder if showing the key structures as a figure in each of the distinct sensor classes would help?
Response to Comment 3: We agree with the reviewer that it can be tedious to move back and forth to visualize the structures. We have decided to include figures of the chemical structures featured in each sensor class at the beginning of their section for clarity (Figures 3-6).

Overall, I strongly recommend publication in RSC Chem. Biol. In addition to the points above, the authors may wish to consider the following in any minor revisions.
Comment 4: Pg 4: Multisite phosphorylation patterns have “been” shown to be functionally significant beyond the capability of single site modification
Response to Comment 4: The word “been” has been added to the sentence.

Comment 5: Pg 5: Additionally, the MS/MS approach is labour intensive and requires costly instrumentation and technical expertise effectively repeats an earlier sentence.
Reponse to Comment 5: The sentence has been removed.

Comment 6: Pg 5: This comprehensive review will cover optical sensing – I would remove “comprehensive”
Response to Comment 6: The word “comprehensive” has been removed.

Comment 7: Pg 9: I suggest rewording of “and hydrophobic interactions of the BODIPY core” to e.g. “and favourable hydrophobic packing of the….”; hydrophobic interactions do not exist, rather the effect that is observed arises as a result of favorable/ unfavorable non-covalent interactions between solute(s) and solvent, so is a solvophobic effect. (repeated on page 17)
Response to Comment 7: We have reworded both mentions of “hydrophobic interactions” with “hydrophobic packing” on page 8, line 27 and page 18, line 7.

Comment 8: In the opening section describing largely the work of Hamachi and focussing on different spacings e.g. i, i + 7 – it would be pertinent to comment on the context recognition of such a cluster presumably has secondary structure context and so there are presumably caveats with the use of peptide based models of full length proteins? (although admittedly for e.g. IDPs and aggregated peptides with regular spacing of phosphosites this is perhaps less pertinent). Alternatively this might work as well in the conclusion as an area for further exploration.
Response to Comment 8: We thank the reviewer for this comment. We have added remarks on use of peptide models and caveats regarding secondary structure of i,i+n residues in the conclusions section. We have added these remarks to the conclusion section to retain the flow of the discussion paragraphs pertaining to each sensor design and application. Additional sentences regarding secondary structure context and peptide-based models have been added to page 26, lines 3 - 9.

Additional changes:
• Page numbers and line numbers have been added to the manuscript which were referred to in the responses to the reviewers.
• The abbreviation for circular dichroism (CD) spectroscopy has been added to page 7, line 11.
• Minor typographical errors have been addressed across the manuscript.
• A typographical error on page 12, line 17 which mislabelled sensor 8 as sensor 7 was changed.
• An Acknowledgements section has been added after the Summary and Perspective section.

19-Apr-2021

Dear Dr Gunning:

Manuscript ID: CB-REV-03-2021-000055.R1
TITLE: Optical Chemosensors for the Detection of Proximally Phosphorylated Peptides and Proteins

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made on the basis of my own evaluation, I am pleased to accept your manuscript for publication in its current form.

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With best wishes,

Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea