Investigation of Ethanol and Isopropanol as Greener Alternatives to Acetonitrile in the RP-HPLC Purification of Histone Tail Peptides Bearing Acylation-Type Post-Translational Modifications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses an important topic in the field of peptide synthesis and purification by exploring sustainable alternatives in high-performance liquid chromatography (HPLC) for histone tail peptides bearing specific post-translational modifications (PTMs). Histone PTMs are crucial regulators of chromatin dynamics and gene expression, and access to highly purified synthetic peptides is essential for structural and epigenetic studies. The authors successfully synthesized histone H4 tail peptides with site-specific lysine modifications using solid-phase peptide synthesis (SPPS) and developed an improved HPLC method capable of separating peptides differing by a single modification. The novelty lies in their evaluation of ethanol and isopropanol as environmentally friendly alternatives to acetonitrile in the mobile phase, aligning with green chemistry principles. The methodology is well described, and the results are clearly presented with convincing evidence that the optimized acetonitrile-based method provides effective resolution suitable for downstream applications requiring high purity. However, the manuscript would benefit from a more detailed comparison of chromatographic performance between acetonitrile and the proposed green solvents, particularly regarding resolution, peak shape, and sensitivity. Additionally, mechanistic insights into why ethanol and isopropanol may or may not replicate acetonitrile’s behavior would strengthen the discussion. The study’s significance is high because it bridges analytical chemistry, sustainability, and epigenetics, though further experiments are needed to fully validate the green solvent approach for high-precision purification. The English is generally clear but could be slightly improved for conciseness. Overall, this manuscript provides a promising step toward sustainable peptide purification and should be considered for publication after minor revisions addressing the comparative data and mechanistic explanations.

Questions

1. What specific molecular interactions explain the difference in peptide separation efficiency between acetonitrile and ethanol/isopropanol?
2. How do the polarity and hydrogen bonding properties of ethanol and isopropanol affect the retention behavior of histone PTM peptides?
3. Could differences in solvent viscosity contribute to variations in peak resolution and backpressure in the HPLC system?
4. Are there any interactions between the mobile phase and the stationary phase that could uniquely impact the behavior of lysine-modified histone peptides?
5. How might the presence of multiple PTMs on a single peptide influence separation efficiency under green solvent conditions?

 

Comments on the Quality of English Language

The English is generally clear and understandable

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

• The manuscript requires thorough English editing. Many sentences are too long and should be broken into shorter, clearer statements. Verb tenses must be checked consistently, especially in the Results section which should be written in the past tense (e.g., line 143). Spelling errors should be corrected (line 151: “separetly” → “separately”).
• The peptide synthesis and modifications are described in the Methods, but there is no mass spectrometry validation. This is a major weakness. Simple MALDI-TOF, LC-MS, or ideally MS/MS should be provided to confirm correct synthesis and incorporation of site-specific PTMs. Chromatograms alone are insufficient for validation. Since the focus is on PTM-bearing histone peptides, MS data are essential for scientific credibility.
• The title currently emphasizes “green chemistry,” but only ethanol and isopropanol were tested as alternatives to acetonitrile. Either the title should be modified to reflect the actual data, or the green chemistry component should be expanded.
• The main conclusion is that acetonitrile remains the most effective solvent, which is already well known in the field. This limits novelty. Including MS-based validation would strengthen the manuscript and improve its scientific contribution.
• The discussion section should be expanded with mechanistic reasoning on why ethanol and isopropanol failed to achieve separation. Future perspectives should be elaborated, such as testing other greener solvents, alternative chromatographic columns, or integrating MS validation with purification strategies. Cite more references in discussion section.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Two histone H4 peptides containing different post-translational modifications (acetylation and paltoacylation) were synthesized. An efficient HPLC purification method was developed, and the feasibility of green chemical solvents ethanol and isopropanol as alternative solvents for acetonitrile was evaluated. However, the experimental results of green solvents failed to achieve the expected separation effect, and the innovation is not fully demonstrated. It is recommended to supplement more solvent screening data or other green chemistry strategies.

The experimental design is generally reasonable, but has the following issues: Too narrow solvent screening range (only three solvents)；Lack of optimization design for column type and temperature；No consideration of industrial scale-up feasibility (e.g., flow rate too low)

Recommendations: (1) Add tests with other green solvents (e.g., acetone, dimethyl carbonate); (2) Compare different columns; (3) Include industrial feasibility analysis.

Shortcomings in experimental methods description: Peptide synthesis method description is too brief, lacking key parameters (e.g., coupling efficiency, crude peptide purity); HPLC condition optimization process is incomplete, lacking systematic method development data; Green solvent experiments did not fully utilize existing equipment conditions (e.g., no heating effect test).

Figure 2 shows effective acetonitrile method, but green solvent part (Figures 3-4) data fail to support "green chemistry" claim.

Lack of quantitative data comparing separation efficiency of different solvent systems;

Insufficient MS data to confirm product purity.

It is suggested to add the following contents: (1) Add quantitative comparison data for green solvents; (2) More MS verification; (3) Possible environmental impact assessment.

Discussion section should more objectively analyze reasons for green solvent experiment failure; Supplement discussion on industrial application prospects.

Improve figure quality (e.g., resolution, label clarity).

Update references (insufficient recent 3-year relevant literature).

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have substantially improved the manuscript and provided thorough and scientifically sound responses to the reviewers' comments. Their revised version includes a more detailed chromatographic comparison and expanded discussion on the solvent effects, thereby enhancing the clarity and rigor of the study. The experimental design is robust, and the interpretation of results is generally well-justified. However, two mechanistic points require further clarification: Why do ethanol and isopropanol fail to induce sufficient differential retention for separating peptides that differ by a single post-translational modification, despite their comparable polarity to acetonitrile? Why does the increased hydrogen bonding capacity of these protic solvents not enhance selectivity for lysine-modified peptides, which presumably interact with the mobile phase via similar mechanisms?

Comments on the Quality of English Language

The English is generally clear and scientifically appropriate, though some sentences could benefit from improved conciseness and structure.

Author Response

Comment: The authors have substantially improved the manuscript and provided thorough and scientifically sound responses to the reviewers' comments. Their revised version includes a more detailed chromatographic comparison and expanded discussion on the solvent effects, thereby enhancing the clarity and rigor of the study. The experimental design is robust, and the interpretation of results is generally well-justified. However, two mechanistic points require further clarification: Why do ethanol and isopropanol fail to induce sufficient differential retention for separating peptides that differ by a single post-translational modification, despite their comparable polarity to acetonitrile? Why does the increased hydrogen bonding capacity of these protic solvents not enhance selectivity for lysine-modified peptides, which presumably interact with the mobile phase via similar mechanisms?

Response: Following Reviewer 1 comments in round 1 we had developed our discussion on the mechanistic reasoning of chromatograms obtained with different solvents. To emphasize even more the effect of hydrogen bonding capacity in our study’s system we added to Discussion as highlighted in green three more sentences.

(1) line 290-291: "Additionally, compared to acetonitrile, ethanol and isopropanol should have less interactions with the C18 column."

(2) line 294-297: "Similarly, the protic solvents’ weaker affinity towards the C18 hydrophobic stationary phase may lead to heterogeneous interactions between the solvent, column and peptides. Achieving a more homogeneous distribution between the mobile and stationary phases could improve the separation of similar peptides."

(3) line 299-301: "The addition of acylation on lysine residues remove their charge. It would be more suitable to have significant charge differences between the peptides to separate when working with protic solvents."

 

Reviewer 2 Report

Comments and Suggestions for Authors

Please revise abstract to include information on the MS identification of the synthesized peptides.

 

Line 68 - The statement “To the best of our knowledge there are no research papers specifically focusing on the purification of PTM bearing histone peptides” needs to be modified. Several related studies exist, so please rephrase to reflect that while very few papers directly address this aspect, the area is not completely unexplored.

 

Move the MS data into the main figure. Consider combining them into a single panel to clearly demonstrate that the synthesized peptides were confirmed by MS. Please annotate the spectra appropriately.

 

Table 4 -Abbreviations (N, H, Tf, etc.) should be defined in the figure legend for clarity.

Author Response

Comment 1: Please revise abstract to include information on the MS identification of the synthesized peptides.

Response 1: The abstract has been updated to inform about the use of MS for synthesis validation.
The following sentence has been added to the Methods part (line 20-21) of the Abstract: "The correct synthesis of the peptides were confirmed by their molecular weights using a mass spectrometer."

Comment 2: Line 68 - The statement “To the best of our knowledge there are no research papers specifically focusing on the purification of PTM bearing histone peptides” needs to be modified. Several related studies exist, so please rephrase to reflect that while very few papers directly address this aspect, the area is not completely unexplored.

Response 2: Although some papers do describe PTM bearing histone peptides we have not encountered any focusing specifically on their purification and the separation of closely related modifications. Therefore we would be grateful if Reviewer 2 could mention us the studies they are referring to so that we could cite them in our manuscript.
In the meanwhile we have modified the sentence to point out that the study of PTM bearing peptide purification has not been extensively elaborated in the literature.
“The study of PTM bearing peptide purification has not been extensively elaborated in the literature”.

Comment 3: Move the MS data into the main figure. Consider combining them into a single panel to clearly demonstrate that the synthesized peptides were confirmed by MS. Please annotate the spectra appropriately.

Response 3: If found convenient by Reviewer 2 we would like to keep the MS figures just in the supplementary file to facilitate our manuscript’s reading. The panel dimensions are too small to merge all data in a single figure. Additionally we would like the main figure of the manuscript to be the chromatogram profiles. However we agree that mentioning the necessity to validate peptide synthesis by mass spectrometry is crucial that is why we had added the calculated peptide fragments (Table 3) in our main text.  Now we have also added the found values on the same table.

Comment 4: Table 4 -Abbreviations (N, H, Tf, etc.) should be defined in the figure legend for clarity.

Response 4: A footnote has been added to the table to guide readers to HPLC performance parameters definitions in Materials and Methods.

Reviewer 3 Report

Comments and Suggestions for Authors

The quality of the manuscript has been improved after revision.

Author Response

We thank Reviewer 3 for helping improve our manuscript via their comments and suggestions.