A general fluorescent light-up probe for staining and quantifying protein

Proteins are the primary functional agents in all cellular processes, facilitating various functions such as enzymes and structure-forming or signal-transducing molecules. In this work, we report a fluorescent dye, PyMDI-Zn, which could specifically bind with proteins and provide a red-shifted fluorescent emission. The visual analysis of protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be realized in 5 min by using PyMDI-Zn as a light-up dye. Based on its cell penetration and low toxicity, PyMDI-Zn could also be applied to locate protein-rich regions and organelles in live cell imaging. Moreover, the direct protein quantitation can be realized based on PyMDI-Zn, providing a method of screening for food adulteration by nitrogen-rich compounds.


Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The authors reported a new function of an interesting compound (PyMDI-Zn). PyMDI-Zn can be used as the fluorescent dye for detecting proteins. In general, the main findings of their work are fairly interesting. However, I am afraid that the manuscript is not carefully prepared and revisions must be needed to improve the work.
The main concern about the manuscript. As stated in the title and the whole manuscript, PyMDI-Zn can be used as staining and quantifying proteins. The works for staining proteins is sound. However, the proof for quantifying proteins is insufficient. The authors provided a calibration curve to show the linearity of the method, and a result showed that fluorescent intensities of protein-PyMDI-Zn are not influenced by urea or melamine. These works are necessary, but not enough. At least they should provide one application of this method for quantifying the protein concentration, to show that the method they proposed really works well when quantifying proteins. Samples such as milk with and without urea/melamine might be appropriate. It should be easy to carry out such experiments since they have already constructed the calibration curve. Traditional method for quantifying protein concentration may be included as control to prove the accuracy of their method. Kjeldahl or Dumas methods could be included as well to show that their method is better. 6. the author stated that the proteins bands in SDS-PAGE could be seen under UV with or without washing step. Thus, it is expected that some images should be provided (at least as supplementary materials) to support the claim. 7. description about the results in table S1 is not clear to me. The table provided the concentration of many substances. What are the responses of protein-PyMDI-Zn to each substances at the given concentration? Any data? 8. there are no descriptions in the method section about how the experiments in part 4.4 are performed.

Is the language acceptable? No
Is it clear how to access all supporting data? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
This paper reports a fluorescent dye, PyMDI-Zn, which could specifically bind proteins and provide a red-shifted fluorescent emission, this study is intersting and provide a useful contribution to the literature. However, some inssues need to be solved before publication. 1) The style of this manuscript is confused, such as Figures.
2)The language needs to be revised and improved.
3)The methods should be compared with other Fluorescent Probe methods,not only CBB.
Decision letter (RSOS-190580.R0) 10-Jun-2019 Dear Dr Tang, The editors assigned to your paper ("A General Fluorescent Light-Up Probe for Staining and Quantifying Protein") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper before 03-Jul-2019. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available, we may invite new reviewers.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Thank you for giving us the opportunity to review your manuscript for publication in Royal Society Open Science. You will see from reviewers' comments copied below that while they find your work of considerable potential interest, they have raised substantial concerns that must be addressed. In light of these comments, we cannot accept the manuscript for publication, but would be prepared to consider a revised version that addresses these concerns.
We hope you will find the referees' comments useful as you decide how to proceed. The major points raised by the Reviewers would need to be addressed if you wished to resubmit a new version of this manuscript. In particular, we agree with both referees that comparison between the present method with other quantification methods would greatly strengthen your conclusions.
Please bear in mind that we will be reluctant to approach the referees again in the absence of major revisions, and the major revision does not imply the paper will be accepted eventually. If you choose to revise your manuscript taking into account all reviewer and editor comments, please highlight all changes in the manuscript text file.
We are committed to providing a fair and constructive peer-review process. We do not generally allow multiple rounds of revision. Based on the concerns to be addressed, we would suggest 30 days to revise. If you do have any queries or concerns, you are welcome to contact me in the first instance.
Thank you again for allowing Royal Society Open Science to review your work. We look forward to hearing from you soon.
Sincerely, Dr Luning Liu Comments to Author: Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The authors reported a new function of an interesting compound (PyMDI-Zn). PyMDI-Zn can be used as the fluorescent dye for detecting proteins. In general, the main findings of their work are fairly interesting. However, I am afraid that the manuscript is not carefully prepared and revisions must be needed to improve the work.
The main concern about the manuscript. As stated in the title and the whole manuscript, PyMDI-Zn can be used as staining and quantifying proteins. The works for staining proteins is sound. However, the proof for quantifying proteins is insufficient. The authors provided a calibration curve to show the linearity of the method, and a result showed that fluorescent intensities of protein-PyMDI-Zn are not influenced by urea or melamine. These works are necessary, but not enough. At least they should provide one application of this method for quantifying the protein concentration, to show that the method they proposed really works well when quantifying proteins. Samples such as milk with and without urea/melamine might be appropriate. It should be easy to carry out such experiments since they have already constructed the calibration curve. Traditional method for quantifying protein concentration may be included as control to prove the accuracy of their method. Kjeldahl or Dumas methods could be included as well to show that their method is better. Reviewer: 2 Comments to the Author(s) This paper reports a fluorescent dye, PyMDI-Zn, which could specifically bind proteins and provide a red-shifted fluorescent emission, this study is intersting and provide a useful contribution to the literature. However, some inssues need to be solved before publication. 1) The style of this manuscript is confused, such as Figures.
2)The language needs to be revised and improved.
3)The methods should be compared with other Fluorescent Probe methods,not only CBB.

29-Jul-2019
Dear Dr Tang, I am pleased to inform you that your manuscript entitled "A General Fluorescent Light-Up Probe for Staining and Quantifying Protein" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published.

Reply to the Comments by Reviewer 1
We greatly appreciate the Reviewer 1 for his/her positive comments: "The authors reported a new function of an interesting compound (PyMDI-Zn). PyMDI-Zn can be used as the fluorescent dye for detecting protein. In general, the main finding of their work are fairly interesting." Meanwhile, we thank reviewer 1 for the professional suggestions for modification of our manuscript, as well as his/her careful reading to help us find the errors in our manuscript. We have modified the paper accordingly, and our point-by-point replies to the questions are as follows: The main concern about the manuscript Thank the reviewer for his/her professional question. To compare with the existing methods, we adopted the reviewer's opinion to use milk as the sample for quantifying the protein concentration. As shown in Figure S8, we prepare three samples: sample 1: milk powder (labeled with a protein concentration of 18% by the producer); sample 2: milk powder with melamine to make the protein concentration up to 35% (calculated by nitrogen %); sample 3: milk powder with urea to make the protein concentration up to 30% (calculated by nitrogen %). We establish standard curves of quantitative methods of Pierce and PyMDI-Zn using BSA as a standard protein. The Kjeldahl method is a means of determining the nitrogen content of organic and inorganic substances, and the nitrogen content of the protein usually accounts for about 16% of its total mass. Therefore, the total protein content of the sample can be calculated by measuring the nitrogen content of the sample (assume that the nitrogen is all from the protein). And the quantitative results of three milk sample with different methods were illustrated Figures S8 as follows:

Figure S8
These data implied that the Kjeldahl method is likely to be applied by the producer of our milk sample. But this method was easily interfered by nitrogen-rich compounds such as melamine and urea. PyMDI-Zn and Pierce Kit both provided a stable data which was not interfered by melamine and urea. Moreover, the protein concentration measured by PyMDI-Zn is closer to that of the Kjeldahl method than Pierce Kit.

Question 1.
The x-or y-axis titles are missing in some of the figures such as fig 1A and 1B. We have added x-or y-axis titles to Figure 1A and Figure 1B accordingly. Thank the reviewer for his/her professional suggestion, and we have added the data of Glucose, Glycogen, and Starch into Figure 1C. There were no significant impacts to the fluorescence of protein-PyMDI-Zn by those substrates, showing the high specificity of PyMDI-Zn probe to protein.  Thank the reviewer for his/her helpful suggestions. We have changed "6times" to "6 times" in part 4.1 accordingly.

Question 4.
fig 2, the same figures appeared in the supplementary materials (fig S2, S3, S4). Repeated usage of figures in this paper is not necessary. Moreover, the fig 2B and fig S3 are exactly the same image, which is not acceptable. Fig S2   and fig S4 provided CBB results for control to fig 2A and 2C. CBB staining is necessary for readers to compare the methods. Therefore, it is suggested that the fig S2-4 is removed, and CBB images in fig S2 and fig S4 are moved to   fig 2. We have removed the Figure S2-4 in the supplementary materials, and added the CBB images in Figure S2 and Figure S4 to Figure 2 according to the suggestion of reviewer 1, which is shown as follows. Thank the reviewer for the careful reading. Protein samples were commercial protein markers that contain proteins of 80 kDa (100 ng/μl), 60 kDa (100 ng/μl), 40 kDa (200 ng/μl). 30 kDa (100 ng/μl), and 20 kDa (100 ng/μl). This information also has been added to the figure caption of Figure  2B.

Question 6.
the author stated that the proteins bands in SDS-PAGE could be seen under UV with or without washing step. Thus, it is expected that some images should be provided (at least as supplementary materials) to support the claim.
Thank the reviewer for the professional comment. As shown in the following Figure S2, almost the same gel-staining result was obtained with or without washing step. Therefore, all SDS-PAGE gels stained by PyMDI-Zn in our manuscript did not go through the washing step. And according to the suggestion of the reviewer, the results of experiments in Figure S2 were added into supplementary materials.
In Description about the results in table S1 is not clear to me. The table provided the concentration of many substances. What are the responses of protein-PyMDI-Zn to each substance at the given concentration? Any data?
Thank the reviewer for this professional question. The responses of protein-PyMDI-Zn to various substances were tested to investigate their interference to the protein assay (J. AM. CHEM. SOC. 2005, 127, 17799-17802). These reactions were carried out in the solution with 100 μM BSA as well as 200 μM PyMDI-Zn in presence of a different concentration of foreign substances. The fluorescence was detected at 520nm with excitation at 486 nm by Thermo Scientific Varioskan Flash. The maximum concentrations that give the perturbation of fluorescence intensity less than 10% can be obtained. We use glucose as an example to illustrate how we get the corresponding concentration ( Figure S3): The fluorescence of protein-PyMDI-Zn with different concentration of glucose was recorded, and the maximum concentration of glucose the give the perturbation of fluorescence intensity less than 10% was about 20 mM. Thank the reviewer for the helpful suggestions. The following are the descriptions of how the experiments in part 4.4 are performed: "100μl solutions containing different amounts of protein (0 to 600 μg) with 100 μM PyMDI-Zn were incubated at room temperature for about 5 minutes. Then, the fluorescence of those samples was detected at 520nm with excitation at 486 nm by Thermo Scientific Varioskan Flash. The concentration of protein was plotted against the corresponding fluorescent intensity to obtain a standard curve. The protein concentration of the unknown samples was determined by the standard curve. Protein solutions are normally assayed in triplicate." We have added this description in part 3.6 into the Materials and Methods section in our revised manuscript.

Reply to the Comments by Reviewer 2
We sincerely appreciate the Reviewer 2 for his/her positive and professional comments. The Reviewer 2 pointed out that: "This study is interesting and provide a useful contribution to the literature." We thank reviewer 2 for his/her useful suggestion to help us improve the manuscript. Following are our point-by-point answers to the comments and suggestions to our manuscript.

Question 1.
The style of this manuscript is confused, such as Figures.
Thank for the helpful suggestion of the reviewer. We have unified the style of our manuscript and supplementary materials.

Question 2.
The language needs to be revised and improved.
Thank the reviewer for his/her careful reading. Our manuscript has been edited by a native English speaker. The manuscript and Supplementary material with correction traces are submitted too.

Question 3.
The methods should be compared with other fluorescent probe methods, not only CBB.

Figure S4
As shown in Figure S4, SYPRO Orange afforded a lower background and a higher fluorescent response to protein bands (50-120 kDa) than PyMDI-Zn on the gel. But, as for the proteins with lower molecular weight (20, 30 and 40 kDa), PyMDI-Zn gave better staining results. And according to the suggestion of the reviewer, Figure S3 have been added into supplementary materials. The main concern about the manuscript.
As stated in the title and the whole manuscript, PyMDI-Zn can be used as staining and quantifying proteins. The works for staining proteins is sound. However, the proof for quantifying proteins is insufficient. The authors provided a calibration curve to show the linearity of the method, and a result showed that fluorescent intensities of protein-PyMDI-Zn are not influenced by urea or melamine. These works are necessary, but not enough. At least they should provide one application of this method for quantifying the protein concentration, to show that the method they proposed really works well when quantifying proteins. Samples such as milk with and without urea/melamine might be appropriate. It should be easy to carry out such experiments since they have already constructed the calibration curve. Traditional method for quantifying protein concentration may be included as control to prove the accuracy of their method. Kjeldahl or Dumas methods could be included as well to show that their method is better.  This paper reports a fluorescent dye, PyMDI-Zn, which could specifically bind proteins and provide a red-shifted fluorescent emission, this study is intersting and provide a useful contribution to the literature. However, some inssues need to be solved before publication.

1) The style of this manuscript is confused, such as Figures.
2)The language needs to be revised and improved.