Lc—ms-based untargeted metabolomics reveals potential mechanisms of histologic chronic inflammation promoting prostate hyperplasia

Jiale Li; Beiwen Wu; Guorui Fan; Jie Huang; Zhiguo Li; Fenghong Cao

doi:10.1371/journal.pone.0314599

Peer Review History

Original SubmissionMay 14, 2024
17 Oct 2024 Decision Letter - Stanisław Jacek Wroński, Editor PONE-D-24-17979Lc - ms-based untargeted metabolomics reveals potential mechanisms of histologic chronic inflammation promoting prostate hyperplasiaPLOS ONE Dear Dr. Li, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Dec 01 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The authors should address the following issues prior to publication: 1.The authors should provide the full forms and significance of all abbreviations (e.g., PV) to enhance comprehension for general readers. 2.Were there any challenges related to ion suppression or enhancement during the LC-MS analysis, and what strategies were employed to address these effects? 3.Can the authors elaborate on the limitations encountered during the sample preparation process and how they were addressed to ensure the reliability of the LC-MS results? By addressing these points, the manuscript will become more comprehensive and informative, thereby improving clarity and ensuring that the presented innovations are fully understood by the readership. With compliments Stanisław Wroński Academic Editor [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This study reveals that histological chronic inflammation significantly alters prostate metabolic profiles, particularly affecting lipid metabolites such as free fatty acids, ceramides, sphingomyelins, and lysophosphatidylcholine. Utilizing LC-MS-based metabolomics, the research establishes these metabolic changes as key contributors to the pathogenesis of inflammation-induced prostate hyperplasia, offering new insights for prevention and treatment strategies. The manuscript is well-written, and I recommend its publication in PLOS ONE. However, I believe the authors should address the following issues prior to publication: 1.The authors should provide the full forms and significance of all abbreviations (e.g., PV) to enhance comprehension for general readers. 2.Were there any challenges related to ion suppression or enhancement during the LC-MS analysis, and what strategies were employed to address these effects? 3.Can the authors elaborate on the limitations encountered during the sample preparation process and how they were addressed to ensure the reliability of the LC-MS results? By addressing these points, the manuscript will become more comprehensive and informative, thereby improving clarity and ensuring that the presented innovations are fully understood by the readership. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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Revision 1
24 Oct 2024 Author Response Response to reviewers Dear editor and reviewers, Thank you for offering us an opportunity to improve the quality of our submitted manuscript " Lc - ms-based untargeted metabolomics reveals potential mechanisms of histologic chronic inflammation promoting prostate hyperplasia" (ID: PONE-D-24-17979R1). I received constructive and insightful comments. In this revision, we have addressed all of these suggestions. We hope the revised manuscript has now met the publication standard of your journal. We highlighted all the revisions in red colour. Below are our responses to the questions posed by the reviewers. Overall comment 1 The authors should provide the full forms and significance of all abbreviations (e.g., PV) to enhance comprehension for general readers. Response: Thank you for your suggestions. We have carefully and meticulously refined the full names of all abbreviations in this paper. We have red-flagged the changes in the article. Comment 2 Were there any challenges related to ion suppression or enhancement during the LC-MS analysis, and what strategies were employed to address these effects? Response: Thank you very much for your professional question. Ion suppression or enhancement is a manifestation of matrix effect, which does exist during LC-MS analysis. The matrix effect in LC-MS is generally thought to occur because non-volatile components of the matrix compete with the substance to be measured, during ionization on the surface of the droplet, affecting the ionization efficiency at the electrospray interface. These non-volatile matrix components draw the droplets together, preventing them from cleaving into smaller microdroplets. The interfering substances that produce matrix effects fall into two categories. One group is called “endogenous substances”: substances that originate from the analyte itself and remain in the final extract, including salts, strongly polar compounds, surfactants, and lipids, amines, peptides, etc., that are structurally similar to the target compound. The other category is called “exogenous substances”: they do not come from the matrix itself, but from the external environment during the establishment of the method, including plastic and polymer residues, ion-pairing reagents, organic acids, buffer solutions, etc. In this experiment, I mainly take the substances from the matrix itself and retain them in the final extraction solution. In this experiment, I took four main programs to reduce the matrix effect. First, we adopted the better extraction scheme of liquid-liquid extraction for tissue metabolites as tested by want et al. The principle is that nonpolar compounds are more soluble in organic solvents than in water. On the contrary, ionic or polar compounds are more soluble in aqueous solutions than in organic solvents. Ultimately, the polar and nonpolar compounds will be extracted as much as possible. Second, after preexperimentation, we applied a variety of columns (HSS T3, BEH C8, BEH C18, CSH Phenyl-Hexyl) with different chromatographic separation conditions. It was found that the application of the columns and chromatographic conditions in this study had minimal matrix effects. Third, we used leucine enkephalin for mass calibration to reduce mass bias. Fourth, Progenesis QI (Waters, Nonlinear Dynamics, Newcastle, UK) is a better metabolomics data processing software, which has the functions of base; line filtering, peak alignment and deconvolution to reduce the experimental result error caused by matrix effect. Due to the fixed equipment, the replacement of ion source was not carried out in this experiment, and it was not possible to assess the extent of the matrix effect of most compounds on the ion source. These are the measures I took to reduce the matrix effect. Comment 3 Can the authors elaborate on the limitations encountered during the sample preparation process and how they were addressed to ensure the reliability of the LC-MS results? Response: Thanks again for the question from the expert, there are indeed many limitations in the sample preparation process. The main ones are following items. First, sample contamination and sample stability. After the patient underwent prostate enlargement surgery, we collected the prostate tissue in sterile, enzyme-free cryopreservation tubes within 30 minutes and stored it in a -80°C refrigerator in the operating room. The vast majority of metabolites will not be degraded and transformed under ultra-low temperature conditions. Second, loss during sample processing. During sample processing, loss of target metabolites may occur due to the different extraction protocols for metabolites and the time required. Therefore, we performed pre-experimentation beforehand to choose a better extraction protocol and operated on dry ice throughout the process to reduce the loss of metabolites. Third, matrix effect. We ensured that all reagents and materials were of high quality and met the requirements for LC-MS analysis. Plastic devices that could interfere, such as bottles, funnels, beakers, etc., were avoided. Likewise the measures taken in the second expert question. Fourth, strict quality control. In the pre-treatment, the original samples were randomly disrupted between all the different groups to avoid batch effects between the comparison groups. For quality control (QC) and to improve the stability of the system, 10 μL of supernatant was transferred from each sample and mixed as QC, and 18.2 MΩ.cm pure water was used as a blank control group, and the QC samples and the blank control group would be injected at regular intervals (every 5 samples) to minimize the sample residues and to monitor the experimental stability. A blank solvent (Blank) was used to balance the instrument status and clean up the column residual material during the assay to avoid sample residue and contamination buildup problems. Preventive maintenance is achieved by pre-collecting QC mixed matrix samples to prevent situations such as unstable data. [Author Name]: JiaLe Li [Affiliation]: Clinical Medical College, North China University of Science and Technology [Mailing Address]: Tangshan, Hebei, China, 063210 [Phone Number]: +86 18352185307 [Email]: 1654989167@qq.com Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0314599.r002
14 Nov 2024 Decision Letter - Stanisław Jacek Wroński, Editor Lc - ms-based untargeted metabolomics reveals potential mechanisms of histologic chronic inflammation promoting prostate hyperplasia PONE-D-24-17979R1 Dear Dr. Jiale Co We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Stanisław Jacek Wroński, M.D., Ph.D, FEBU Academic Editor PLOS ONE Dear Authors, after careful consideration of revised version (code PONE-D-24-17979R1) and the reviewer's opinion, I conclude that in its present form the article can be approved for publication in POLS ONE. The authors have effectively addressed all concerns raised, resulting in a significantly improved manuscript. With compliments Stanisław Wroński Academic Editor https://doi.org/10.1371/journal.pone.0314599.r003
Formally Accepted
15 Nov 2024 Acceptance Letter - Stanisław Jacek Wroński, Editor PONE-D-24-17979R1 PLOS ONE Dear Dr. Li, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Stanisław Jacek Wroński Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0314599.r004

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