Simultaneous Determination of Steroidal Alkaloids and Polyphenol Group from Eight Varieties of Siberian Solanum tuberosum L. through Tandem Mass Spectrometry

Round 1

Reviewer 1 Report

The manuscript is devoted to determination and research steroidal alkaloids and polyphenol group from Solanum tuberosum L., which opens new areas in metabolome analysis of Solanum tuberosum L., but it needs major corrections. Please check the grammar and correct the following remarks:

The text doesn't contain the relevance of the study. It should be added and justify the value or harm of the analyzed metabolites and their significance for potato growing and the food industry. What was the main purpose of amino acids determination and how are they connected to alkaloids and the topic of the manuscript?

Lines 60-65 Please move fig. 1. to the materials and methods chapter so that it corresponds to the type of manuscript.

Line 60. Please explain what influenced the choice of potato varieties. Are they well studied and used in many studies?

Line 67 Rename the chapter “Experimental” into “Materials and Methods” it's common chapter name pattern.

Line 68 Please add short description of the varieties. If you use geographic naming "Siberian" please explain the difference between common potato varieties and Siberian.

Line 68-69 Please Rewrite as “We used 8 varieties …. For our research” or use "Objects of the study were ". Add a photo of tubers and their cuts of all used varieties as in Figure 1.

Line 77-78 Please rewrite “HPLC-grade acetonitrile was used as a mobile phase for determination … (Fisher Scientific, Southborough, UK), MS-grade formic acid (Sigma-Aldrich, Steinheim, Germany) was used for … “.

Line 82 Add what part of the potato was subjected to research above ground or underground. Had this part of the experience statistical repetitions?

Lines 83-85 Were plant parts crushed when extracts were obtained? Please specify.

Lines 90-91 Please add the manufacturer and commercial name of the column.

Lines 102-103 Specify the gas used.

Line 143 Specify units of measurements.

Figure 3-6 Add a description and decoding parameters of the mass spectra to the text (it is not clear what the four columns in the mass spectrum describe and what they divide into).

Line 143 Table 2 should be shortened, it does not correspond to the type of manuscript.

Line 207 Appendix 1. Table 1. It is advisable to add potato varieties where metabolites have already been found instead of the last column, which indicates the plants in which these metabolites are found.

Please provide links to the references in the text. All the references should be mentioned in the introduction discussion of the manuscript.

Author Response

Lines 60-65 Please move fig. 1. to the materials and methods chapter so that it corresponds to the type of manuscript.

Thank you very much for your work on the manuscript and your time. Your instructions have been fulfilled.

Thanks a lot for the explanation. Sample selection is covered in detail in the Introduction and Materials and Methods. Inserted text highlighted in blue.

Line 67 Rename the chapter “Experimental” into “Materials and Methods” it's common chapter name pattern.

This instruction has been fulfilled.

Line 68 Please add short description of the varieties. If you use geographic naming "Siberian" please explain the difference between common potato varieties and Siberian.

Thanks a lot for the explanation. Sample selection is covered in detail in the Introduction and Materials and Methods. Inserted text highlighted in blue.

Line 68-69 Please Rewrite as “We used 8 varieties …. For our research” or use "Objects of the study were ". Add a photo of tubers and their cuts of all used varieties as in Figure 1.

Detailed cultivar descriptions added to Materials and Methods.

Line 77-78 Please rewrite “HPLC-grade acetonitrile was used as a mobile phase for determination … (Fisher Scientific, Southborough, UK), MS-grade formic acid (Sigma-Aldrich, Steinheim, Germany) was used for … “.

This instruction has been fulfilled.

Line 82 Add what part of the potato was subjected to research above ground or underground. Had this part of the experience statistical repetitions?

The study was subjected to potato tubers. Relevant changes have been added to the text. All experiments were triple repetition

Lines 83-85 Were plant parts crushed when extracts were obtained? Please specify.

Ascentis Express C18 (Supelco, Sigma-Aldrich Japan, Tokyo). Relevant changes have been added to the text.

 

Here we are talking about air injection by a compressor for the operation of an ion trap.

 

The table has been completely changed and redone in accordance with the recommendations. The previous table is incorrect.

Figure 3-6 Add a description and decoding parameters of the mass spectra to the text (it is not clear what the four columns in the mass spectrum describe and what they divide into).

Additional explanations are inserted in the text. All additional texts are marked in blue.

Line 143 Table 2 should be shortened, it does not correspond to the type of manuscript.

The table has been completely changed and redone in accordance with the recommendations. The previous table is incorrect.

Line 207 Appendix 1. Table 1. It is advisable to add potato varieties where metabolites have already been found instead of the last column, which indicates the plants in which these metabolites are found.

The identified biologically active substances are described in detail by potato varieties and are presented in Table 2.

 

 

Reviewer 2 Report

The manuscript "Simultaneous determination of steroidal alkaloids and poly- 2 phenol group from eight varieties of Siberian Solanumtu berosum L. through tandem mass spectrometry" was written well. But it still has some problems.

 

Some were listed (but not limited to) in below, authors should check the whole manuscript carefully.

1. “{[M+H]+- 146}” should be checked. The format is not right.

2.  Line 146, 147 “№№ 67-85”. Please check it.

3. “Table 2. №“. Please check it in table 2.

4. The species name was not right. For example, Solanum tuberosum should be S. tuberosum, except initial one.

5. What does the different color mean? please clarify that in your manuscript.

6. In Table 2, it looks these compounds not listed in a reasonable order. Is it possible to sort them in some way to easy understand? Please clarify this point in your manuscript.

7. Figure 2. is not clear to understand. You should give details about the figure. The figure seems not organized well.

8. In addition, it did not clearly tell why your conduct this study in Introduction. What is the significance and necessary for the present study? What’s important innovative findings?

9. Bibliography appeared in an inconsistent style. And there are some errors about format.

Author Response

 

 

The manuscript "Simultaneous determination of steroidal alkaloids and poly- 2 phenol group from eight varieties of Siberian Solanumtu berosum L. through tandem mass spectrometry" was written well. But it still has some problems.

Some were listed (but not limited to) in below, authors should check the whole manuscript carefully.

1. “{[M+H]+- 146}” should be checked. The format is not right.

Thank you very much for your work and the time spent on the article. Found errors corrected in the text.

2. Line 146, 147 “№№ 67-85”. Please check it.

This error has been fixed. Table 2 has also been completely redone. All corrections and additions are highlighted in blue.

3. “Table 2. №“. Please check it in table 2.

Table 2 has also been completely redone. All corrections and additions are highlighted in blue.

4. The species name was not right. For example, Solanum tuberosum should be S. tuberosum, except initial one.

All corrections according to this comment are included in the text.

5. What does the different color mean? please clarify that in your manuscript.

All fixes added and highlighted in blue

6. In Table 2, it looks these compounds not listed in a reasonable order. Is it possible to sort them in some way to easy understand? Please clarify this point in your manuscript.

Table 2 has been completely redone according to the comments.

7. Figure 2. is not clear to understand. You should give details about the figure. The figure seems not organized well.

The interpretation of the ion chromatogram legend is presented. All changes are highlighted in blue.

8. In addition, it did not clearly tell why your conduct this study in Introduction. What is the significance and necessary for the present study? What’s important innovative findings?

Thank you very much for your questions and clarifications. According to these comments, many new changes and additions have been made to the text. Also, all changes are highlighted in blue.

9. Bibliography appeared in an inconsistent style. And there are some errors about format.

All bibliographical errors have been corrected.

 

 

 

 

 

 

 

Reviewer 3 Report

This article identified metabolic components in extracts of eight different species of Solanum tuberosum L. by using HPLC tandem mass spectrometry, mainly polyphenols and glycoalkaloids, in the extracts. Eighty-three target extracts were identified in the extracts, and a number of newly identified metabolic compounds were added. Relevant studies on the abundance of metabolites among the germplasm of Solanum tuberosum L. were added. However, after a careful review of the manuscript, it was found that the article does not provide an adequate summary of the figures and does not clearly indicate the comparative metabolomic analysis of the eight varieties of potato and the association with the reduction of potato nutrient resistance gene varieties mentioned in the introduction. Therefore, I recommend a partial revision prior to possible publication. Specific comments are given below:

Abstract

For two colored potato varieties may be noted.

A sentence about the application and significance of this work could be added at the end.

Keywords

tandem mass spectrometry Suggested substitution or deletion, duplication with title content.

Introduction

Lines 30-34: Suggested additional references for the potato genotype pool situation

Line 38: Are the seven genotypes the same as the eight varieties studied in the article? Please specify which varieties.

Lines 60-61: Only two varieties of potatoes are described and pictured here. It is suggested that the diagrams of the eight potatoes in Figure 1 should be more intuitive or supplemented with text describing the phenology of the other six varieties of potatoes.

Chemicals and Reagents

Line 80: The rest of the material is of analytical purity level stating that it is not entirely accurate and that there are conflicting levels of methanol reagents used in the following extracts.

Fractional maceration

Lines 82-83: The experimental handling of potatoes and sampling should be fully described, whether the potatoes are in a crushed or cut into pieces. This will affect the efficiency of the extraction.

Line 84: The different levels of methanol reagents used for extraction should be described separately in the Chemicals and Reagents section.

Line 87: Please explain in full how the extract was concentrated for the experiment.

Results and discussion

Line 113: Missing the name of a potato variety, please add.

Line 141: Suggest adding an appropriate description or summary language of the contents of Table 2 to highlight the metabolomic differences among the eight varieties.

Conclusions

This paragraph is overly repetitive with the abstract section, the general conclusion of the purpose of the study is not summarized and the relationship between the listed genotypes and the content of the ligand alkaloids and polyphenols is not explained.

Figures and Tables

Figures and tables should be understandable in the absence of text: please give more detailed titles and descriptions, especially to explain what the study is about.

-Table 1: The names of the reference columns should be described completely and should be able to explain the contents of the table.

-Table 2: Some of the font colors are rather similar to the background color, not easy to read, it is suggested that the font color can be adjusted.

-Figure 1: It is recommended that all 8 potatoes in Figure 1 be put on the graph will be more intuitive.

-Figure 3-6: Suggest adjusting and expanding the font size in the picture to make it more clear.

 

Author Response

 

Abstract

For two colored potato varieties may be noted.

A sentence about the application and significance of this work could be added at the end.

Keywords

tandem mass spectrometry Suggested substitution or deletion, duplication with title content.

Thank you very much for your work and your time. Keyword list changed.

Introduction

Lines 30-34: Suggested additional references for the potato genotype pool situation

Thank you very much for your work. The article has been amended in many ways. All amendments are highlighted in blue

Line 38: Are the seven genotypes the same as the eight varieties studied in the article? Please specify which varieties.

All presented varieties according to the instructions are described in detail in the chapter Materials and Methods

Lines 60-61: Only two varieties of potatoes are described and pictured here. It is suggested that the diagrams of the eight potatoes in Figure 1 should be more intuitive or supplemented with text describing the phenology of the other six varieties of potatoes.

All presented varieties according to the instructions are described in detail in the chapter Materials and Methods

Chemicals and Reagents

Line 80: The rest of the material is of analytical purity level stating that it is not entirely accurate and that there are conflicting levels of methanol reagents used in the following extracts.

Checked.

Fractional maceration

Lines 82-83: The experimental handling of potatoes and sampling should be fully described, whether the potatoes are in a crushed or cut into pieces. This will affect the efficiency of the extraction.

Potato tubers were cut into cubes measuring 3 mm by 3 mm, then maceration was carried out according to the article Azmir et al., 2013. Samples of maceration of potato samples were injected into a mass spectrometer and then analyzed with an ion trap.

Line 84: The different levels of methanol reagents used for extraction should be described separately in the Chemicals and Reagents section.

Checked.

Line 87: Please explain in full how the extract was concentrated for the experiment.

The extract was concentrated according to the method described in the article Azmir et al., 2013

Results and discussion

Line 113: Missing the name of a potato variety, please add.

Thank you very much, this bug has been fixed.

Line 141: Suggest adding an appropriate description or summary language of the contents of Table 2 to highlight the metabolomic differences among the eight varieties.

An additional discussion has been added and the text is highlighted in blue.

Conclusions

This paragraph is overly repetitive with the abstract section, the general conclusion of the purpose of the study is not summarized and the relationship between the listed genotypes and the content of the ligand alkaloids and polyphenols is not explained.

All these comments have been taken into account, the tables have been completely redone for easier reading, and additional texts have been inserted.

Figures and Tables

Figures and tables should be understandable in the absence of text: please give more detailed titles and descriptions, especially to explain what the study is about.

-Table 1: The names of the reference columns should be described completely and should be able to explain the contents of the table.

-Table 2: Some of the font colors are rather similar to the background color, not easy to read, it is suggested that the font color can be adjusted.

-Figure 1: It is recommended that all 8 potatoes in Figure 1 be put on the graph will be more intuitive.

-Figure 3-6: Suggest adjusting and expanding the font size in the picture to make it more clear.

 Checked.

 

Round 2

Reviewer 1 Report

The manuscript is devoted to the important topic of determining the biochemical composition of Siberian potato varieties. Authors have done a great work to correct the comments, but the manuscript still needs to be corrected. Pay attention to the following recommendations:

Line 76. At the end of the Introduction chapter, clearly describe in a few sentences the main purpose of the study. Why did you look at these indicators and how cultivars are evaluated according to them.

Line 84-122. Present the description of the cultivars in the form of a table according to the main qualities (flesh color, skin color, application, etc.). This will reduce the amount of text and make the description more structured.

Fig. 1-3. The photos are not of very good quality, especially Fig. 3, and their backgrounds are different. I think they should either be replaced or taken out as additional materials to the manuscript (in a particular file).

Line 103. Results and Discussion (Change)

Lines 140-141. Specify the role of Formic acid in the analyses. For example: “Formic acid is often used as a component of mobile phase in reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and separation techniques for the separation of hydrophobic macromolecules, such as peptides, proteins and more complex structures including intact viruses”.

Lines 202-206. It is better to put this information in an easy way to fig. 4 or in addition to the name of the figure.

Table 2. This table is very informative and valuable, but difficult to read and takes up a lot of space. Perhaps it is worth making it into additional materials, and presenting a shortened version in the text or giving a description of the main significant components. Check its design for compliance with the requirements of the journal.

There is lack of discussion in “Results and Discussion” chapter. Please add an analysis of the results obtained with links to other conducted research (what do you have in common, what is new, what are the prospects for your study).

In the “Conclusions” chapter, you list the identified substances without any explanation of the novelty and value of this study. Please, in this section, present the prospects of research, the possibilities of their application, explain what makes them unique.

Please check the use of articles ("a" and "the") throughout the text.

Check the placement of references. They should be numbered in order as they are mentioned in the text.

Author Response

Line 76. At the end of the Introduction chapter, clearly describe in a few sentences the main purpose of the study. Why did you look at these indicators and how cultivars are evaluated according to them.

Thank you very much for working on the text and pointing out our mistakes and inaccuracies. We presented the studied potato samples in detail in Table 1 with a description of the obvious advantages of each sample.

Line 84-122. Present the description of the cultivars in the form of a table according to the main qualities (flesh color, skin color, application, etc.). This will reduce the amount of text and make the description more structured.

We presented the studied potato samples in detail in Table 1 with a description of the obvious advantages of each sample.

Fig. 1-3. The photos are not of very good quality, especially Fig. 3, and their backgrounds are different. I think they should either be replaced or taken out as additional materials to the manuscript (in a particular file).

We presented the studied potato samples in detail in Table 1 with a description of the obvious advantages of each sample.

Line 103. Results and Discussion (Change)

Necessary changes have been made to the text

Lines 140-141. Specify the role of Formic acid in the analyses. For example: “Formic acid is often used as a component of mobile phase in reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and separation techniques for the separation of hydrophobic macromolecules, such as peptides, proteins and more complex structures including intact viruses”.

Necessary changes have been made to the text

Lines 202-206. It is better to put this information in an easy way to fig. 4 or in addition to the name of the figure.

It seems to us that this figure is necessary to illustrate

Table 2. This table is very informative and valuable, but difficult to read and takes up a lot of space. Perhaps it is worth making it into additional materials, and presenting a shortened version in the text or giving a description of the main significant components. Check its design for compliance with the requirements of the journal.

Yes, you are absolutely right, this table is really very large and we moved it to the Appendix

There is lack of discussion in “Results and Discussion” chapter. Please add an analysis of the results obtained with links to other conducted research (what do you have in common, what is new, what are the prospects for your study).

The discussion is expanded, additional research is done with the help of Venn diagrams.

In the “Conclusions” chapter, you list the identified substances without any explanation of the novelty and value of this study. Please, in this section, present the prospects of research, the possibilities of their application, explain what makes them unique.

Necessary changes have been made to the text

 

Please check the use of articles ("a" and "the") throughout the text.

Check the placement of references. They should be numbered in order as they are mentioned in the text.

Reviewer 2 Report

There are still some minor errors.

The charts were low quality, and should be re-taken.

 

Author Response

There are still some minor errors.

Checked.

 

The charts were low quality, and should be re-taken.

Checked.

Reviewer 3 Report

The article provides a balanced view of the topic, include all of the current applications, ideas, and hypotheses are properly accounted for.

Author Response

The article provides a balanced view of the topic, include all of the current applications, ideas, and hypotheses are properly accounted for.

 

Thank you so much.