Derivatizing Agent Selection for Hydrophilic Lysine- and Arginine-Containing Tetradecapeptide Analysis in Human Plasma by RP HPLC-MS/MS

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled " Derivatizing Agent Selection for Hydrophilic Lysine- and Arginine-Containing Tetradecapeptide Analysis in Human Plasma 3 by RP HPLC-MS/MS by  Tokareva et al. presents a detailed investigation into the selection of derivatizing agents for the analysis of a hydrophilic lysine- and arginine-containing tetradecapeptide (HEP-1) in human plasma using RP HPLC-MS/MS. The manuscript is well-structured and addresses a significant analytical challenge. However, there are areas where the manuscript could be improved for clarity, rigor, and impact.

and impact.

Major Comments

Abstract

• The abstract should succinctly summarize the key findings, including the specific advantages of propionic anhydride over other agents (e.g., reproducibility, yield, sensitivity).
• Mention the practical implications of the study, such as potential applications in pharmacokinetic studies or clinical settings.

Introduction

• The introduction provides a good background but could better highlight the novelty of the study. Emphasize how this work fills gaps in existing literature, particularly for HEP-1 or similar peptides.
• Clarify why HEP-1 was chosen as the model peptide. Is it representative of a broader class of peptides, or does it have unique properties?

Results and Discussion

Section 2.1 (Peptide Therapeutic to Study)

• The biological relevance of HEP-1 is mentioned, but its structural and functional uniqueness should be elaborated. Why is its analysis particularly challenging?

Section 2.2 (Mass Spectrum)

• The absence of single-charged ions is noted, but the explanation is brief. Expand on why multi-charged ions are more favorable for fragmentation.

Section 2.3 (Derivatizing Agent Selection)

• The rationale for selecting the eight derivatizing agents could be clearer. Were they chosen based on prior success with similar peptides?
• Include a table or flowchart early in the section to summarize the evaluation criteria (e.g., fragmentation efficiency, yield, reproducibility) for each agent.
• The statement that arginine residues are unlikely to react under the conditions used should be supported by references or experimental evidence.

Section 2.4 (Sample Preparation Selection):

1) The observation that HEP-1 co-precipitates with plasma proteins is intriguing but speculative. Provide data or references to support this hypothesis.

• The side reaction with methanol is noted, but the mechanism is not explored. A brief discussion or citation would strengthen this section.

Figures and Tables:

• Figure 1:The peptide structure is helpful, but the labels for acidic and basic sites are unclear. Use distinct colors or symbols for better clarity.
• Figure 3:The mass spectra for propionylated HEP-1 are critical but poorly described. Label key peaks and fragmentation patterns to aid interpretation.
• Table 2:This table is useful but could be expanded to include quantitative metrics (e.g., signal intensity, retention time) for each agent.
• Table 3:The sample preparation scheme is detailed but could be simplified for readability. Consider using a flowchart instead.

Conclusion:

• The conclusion should explicitly state the limitations of the study (e.g., the need for further validation in clinical samples, potential interference from plasma components).
• Highlight future directions, such as applying this method to other hydrophilic peptides or optimizing derivatization for arginine residues.

Materials and Methods:

Section 4.4.4 (Propionic Anhydride Derivatization):

1) The derivatization procedure is complex. Include a step-by-step protocol in the supplementary material for reproducibility.

2) Clarify the rationale for using propionic acid in methanol for protein precipitation. Is this a common practice, or was it optimized in this study?

Minor Comments:

Language and Clarity:

• The manuscript contains some grammatical errors and awkward phrasing. For example:
• "The findings revealed propionic anhydride as the most effective derivatizing agent, enabling high and reproducible product yield that demonstrate sufficient retention on RP column and easily detectable in MRM mode."

Suggested revision: "The findings revealed propionic anhydride as the most effective derivatizing agent, enabling high and reproducible product yields, sufficient retention on the RP column, and easy detection in MRM mode."

• Avoid passive voice where possible (e.g., "It was hypothesized" → "We hypothesized").

Abbreviations:

• Define all abbreviations at first use (e.g., HILIC, ICAT, ICPL).
• Ensure consistency in abbreviation usage (e.g., "RP-HPLC-MS" vs. "RP HPLC-MS/MS").

Supplementary Material:

• Mention the availability of supplementary figures (e.g., mass spectra for other derivatizing agents) in the main text. These are critical for evaluating the study's comprehensiveness.

 

Suggestions for Improvement:

1. Quantitative Data:
• Include statistical analysis or numerical comparisons (e.g., signal-to-noise ratios, recovery rates) to strengthen the conclusions about propionic anhydride's superiority.
2. Mechanistic Insights:
• Discuss why propionic anhydride outperforms other agents. Is it due to steric effects, reactivity, or compatibility with MS detection?
3. Validation:
• Briefly describe the validation parameters (e.g., LOD, LOQ, linearity) in the results section rather than only mentioning them in the methods.

 

Final Recommendation:

The manuscript presents valuable research with practical implications for peptide analysis. However, it requires revisions to improve clarity, rigor, and impact. Addressing the above comments will enhance its suitability for publication.

Decision: Major revision required

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript ID: analytica-3683322 entitled “Derivatizing Agent Selection for Hydrophilic Lysine- and Arginine-Containing Tetradecapeptide Analysis in Human Plasma by RP HPLC-MS/MS“ by Tokareva et al. can be accepted to publication in Analytica after minor revision.

 

Specific comments  

   

According to the Authors, the aim of the study was to provide a step-by-step derivatizing agent selection for hydrophilic lysine- and arginine-containing human ezrin peptide-1 (HEP-1) for further analysis by HPLC-MS/MS in human plasma. Thus, various derivatizing agents (8) were tested to obtain quadruply substituted product with good MS/MS fragmentation and sensitivity. Among the tested agents, propionic anhydride offered the best results. Additionally, different sample preparation protocols of human blood plasma samples were investigated. Among them, protein precipitation with a propionic acid solution in methanol was selected as the most effective method. According to the authors, the study allowed to indicate the potential of propionic anhydride as an effective agent for enhancing the detectability of HEP-1 and its potential suitability for other hydrophilic peptides containing lysine and arginine in analyses of complex biological samples. In my opinion, the obtained results can be interesting for the readers, and the manuscript can be published in Analytica after minor revision.

 

1. The legends to Figures reported in Supplementary material should be added.
2. In the manuscript there are editorial mistakes e.g.:

• The number of Figures included in Supplementary material were not correctly included to the manuscript e.g. Page 5 line 167; (Table 1A, Figure Error! Reference source not found.S1).
• Other editorial mistakes:

Page 2 lines 44, 57  and 80; mass-spectrometry, doses[12], byproducts

                        Page 3 line 116; (Table 1,).

                        Page 8 Table 3 and other parts of the manuscript: µl or ml

Page 9 line 263; byproducts.

These mistakes should be corrected.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper is studying eight derivatizing reagents used for a chosen tetradecapeptide (denoted by HEP-1) in the view of LC-MS/MS determination from human plasma matrix.  LC separation was carried out on a C18 column, under RP conditions, and detection relied on electrospray ionization interface with positive mode.  Although a previous study published by authors already applied the best claimed of these derivatization procedures (i.e. with propionic anhydride, see ref. [52]), this study enlarges this complex problem to other possibilities and finds valuable results about this topic.  However, some additional discussions should be added to the present version, in order to emphasize the importance of such study and clarify some issues.  The main comments refer to the following aspects of the analytical research applied to the derivatization procedures:

1. Separation results have to be more detailed and compared, such as the retention time values for derivatized and underivatized HEP-1, because the attachment of the propionic (or other) moiety are seriously modifying the interaction property of studied molecule with stationary phase. Also here, some discussion on selectivity towards potential interference from the plasma matrix (see line 84) is necessary for practice.
2. This aspect should include the two discussed forms of derivatized HEP-1; the authors advance some discussion about MS response on 3- and 4-substituted with propionyl of HEP-1, but a difference between their retention should have been noticed.
3. The concentration levels of HEP-1 that are expected to be determined from biomatrix are useful from pharmacokinetics point of view (as only briefly mentioned in lines 109-111). In lines 270-276 some previous data on this derivatization are briefly shown, but it will be useful for give information on the detection limit, with and without derivatization, such that to emphasize the performance of this approach in potential applications.
4. To the conditions presented in section 4.2, column temperature and injection volume should be included, and specified if they were the same for all studied derivatization procedures applied in this study or modified according to a certain purpose.
5. The necessity of using an internal standard is recommended and discussed in a new version.
6. The complexity of derivatization with propionic anhydride (section 4.4.4) compared to other possibilities (mainly with benzoic anhydride, section 4.4.3) is somehow not favorable to the choice of the first one, which is relying on more steps, including extraction and evaporation, which are tedious and errors affected. This aspect should be taken into account when concluding the choice of the proposed method to some real applications.  
7. However, the purposes of derivatization in proteomics analysis are correctly enlisted (lines 71-88), but this does not result for the present study: does this proposed method improve the ionization efficiency in ESI interface or improve the chromatographic separation of target analyte towards many interferences from biomatrix ?  These questions should be argued. 

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The new version brought many improvements and added new information and discussions (tables and figures) on the experimental results.  Yes, new Scheme 1 is very illustrative and useful for understanding the entire derivatization procedure.  Agree with the answers of authors from their letter to the observations from the previous reviewing report.  Concluding, the new version can be published in its current form in this journal.