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Since all critiques were adequately addressed and the manuscript was amended accordingly, I am please to accept the revised version.
[# PeerJ Staff Note - this decision was reviewed and approved by Valeria Souza, a PeerJ Section Editor covering this Section #]
The authors addressed my concerns.
I would suggest the authors change "1065 endogenous peptides" into "1040 endogenous peptides" in the abstract and introduction, which is more in line with the results.
The authors addressed my concerns.
The authors addressed my concerns.
The authors addressed my concerns.
Please address the concerns of the reviewers and amend your manuscript accordingly.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the response letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the response letter. Directions on how to prepare a response letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
This manuscript utilizes LC-MS approach to profile endogenous peptides of MRSA and MSSA, aiming to provide a guidance for further mechanistic studies and treatments of S. aureus. They identified 292 MSSA-abundant endogenous peptides and 35 MRSA-abundant endogenous peptides with distinct potential characteristics. The manuscript is well organized and written. However, there are certain gaps and misleading parts in this manuscript, which need to be improved. The idea behind this manuscript is clear but still in early stages.
Concerns:
1. Introduction: this part is not well introduced, which couldn’t clearly express the purpose of this study. Why the authors preferred using peptidome approach, what’s the advantage of it. Are there published literatures talking about the importance of peptides in S. aureus, and their relationship with human diseases?
2. The resolution/quality of all the figures should be improved.
3. The authors should convert the supplementary files into more general formats, like excel.
Sample preparation:
1. the authors boiled the samples for 15 mins to eliminate proteolytic activity. Will this step cause the denaturation of proteins or peptide or other artifacts?
2. Why not use any proteases inhibitors during sample processing?
3. The authors used a 30-kDa molecular weight cut-off filter to enrich the endogenous peptides, however, the supernatant may still contain other components, like protein, nucleic acid. Did the author carry out further steps to enrich the endogenous peptides before submitting to MS analysis? Why used a 30-kDa filter, which is much bigger than a typical peptide, there should be smaller cut-off filters.
Overall, this study presents descriptive data without any functional validation.
1. line 145, “A total of 1040 endogenous peptides from 282 proteins were identified”. I think the authors may mix the concepts between peptide and protein. When working with MS, usually the proteins will be digested into short peptides, and then matched back to identify the original protein. So here, the authors actually identified 282 proteins, not the real endogenous peptides. Even though the author could claim the peptides are generated by the 282 precursor proteins, how could the author make sure that all the peptides are naturally existing in S. aureus, not introduced by sample processing, like the procedure of boiling the sample for 15 mins. If so, the original purpose of this study should be questioned. Please explain it.
2. Also, in the abstract, the author mentioned they identified 1065 endogenous peptides, so what are the rest 25 peptides? (1065-1040=25).
The manuscript is well structured and written clearly with description of sufficient background.
Tu et al., compared endogenous peptidome profile of Methicillin-resistant S. aureus (MRSA) with the methicillin-susceptible S. aureus (MSSA) following appropriate experimental design including sample selection and experimental workflow as well as data analysis.
Tu et al., identified 1065 endogenous peptides from 282 proteins and further analyzed differentially expressed peptide features and precursor protein employing various bioinformatic tools.
The following minor comments may need to be addressed
1. The MSSA has enriched motif but not for MRSA (Figure 3) which may be due to small number of identified peptides expressed in MRSA compared to MSSA. So, the authors may comment the need for deeper peptide coverage.
2. The authors may add more description in bioinformatics criteria including motif-x (line 203), normalization method or if any quantitative imputation is employed (Table S2).
3. The authors may provide the raw data and MaxQuant search result (txt folder) accessible for the community in addition to the identification result provided in supplementary table.
Overall, the study is well designed and written which may provide insight for further in-depth study of S. aureus peptidome and I recommend the manuscript for publication.
There are some minor grammatical issues that needs to be rectified.
1) In the Abstract, line 26-27, Instead of writing "MSSA-abundant endogenous peptides have significantly enriched motif of VXXXK at the C-terminus", you can use "MSSA-abundant endogenous peptides have significantly enriched VXXXK motif at the C-terminus".
2) In the Introduction, line 34-35, Instead of writing "Gram-positive bacterium that is a common cause of human infections", you can use "Gram-positive bacterium that is a common cause of infections in humans"
3) Line 90, remove "the" from the sentence " the acetic acid was then to the lysate.
4) Line 159, remove "s" from lengths in the sentence "relatively short in lengths".
5) Throughout the manuscript, please use italics, whenever the name of the bacteria or the name of a gene is mentioned.
6) Space is missing at several places in the paragraphs when a reference has been inserted in brackets. For example: Line 47 "colonies(Akil and Muhlebach, 2018), Line 48 "thickened cell walls(Garcia et al., 2017) etc.
no comment
1) In the Results section, Lines 166-167, simply because most of the analyzed peptides are acidic and have similar size, it cannot be generalized that they have similar properties. The sentence needs to be rephrased to make more sense.
2) In the Results section, Lines 223-224, "indicating that many precursor proteins were involved in ribosome or translational regulation". This sentence is not clear since ribosomes are the major components of translation. Does the authors mean "ribosome biogenesis ?". This is based on the fact that 13 peptides were found to be structural components of the ribosome.
3) In the Discussion, Lines 252-254, "MRSA-AEP may experience.........regulated by proteases". This statement is not clear and needs to be elaborated for a better understanding of the authors intensions.
4) In the Discussion, Lines 273-275, “In comparison with MRSA-AEP, MSSA-AEP has stronger ribosome related functions including promoting bacterial growth and resistance to antibiotics”. It is acceptable that MSSA-AEP has stronger ribosome related functions like promoting bacterial growth, but the authors does not show any evidence to state that it promotes resistance to antibiotics, though ribosome are targeted by a number of antibiotics.
Overall, the manuscript is well written and the role of endogenous peptides in the regulation of bacterial growth and immune responses were well explained. There are some minor issues in the results and discussion section that needs to be clarified in order to improve the quality of the manuscript. I recommend this article for publication.
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