1. Author Response:

    Evaluation Summary:

    This well-done study establishes a work flow for the analysis of the peptidome of wound fluids. By doing so it enables the identification of peptide patterns associated with wounds that are healing versus non-healing. The method may therefore help to define candidate biomarkers for wound healing. Overall enthusiasm was somewhat dampened by findings previously reported by the same group and also by others.

    We appreciate the positive evaluation of the work and its applicability and have now added clarifications and information on the relationship between this and the subsequent Frontiers Immunology paper published by our group. We want to stress that the current eLife MS was originally uploaded on the preprint server medRxiv on the 3rd of November 2020. The Frontiers paper, which is a follow up study of the current manuscript, was published in February 2021. Importantly, the latter is based on, and refers to, the original methodology and peptidome data described in the medRxiv article (which was then later transferred to eLife). In the current revised version of the manuscript, we now clearly describe the originality of the methodology described here and its precedence, and the overall separate and independent character of the current manuscript. In particular, we now thoroughly discuss the findings of this present study in relation to the Frontiers article and therefore, we believe that the uniqueness of the present paper is now made very clear. Therefore, in our opinion, the Frontiers article does not diminish the novelty and strength of the current manuscript, as it has an overall separate and independent character. Instead, it increases its strength as we showed that the here described method and obtained qualitative results can be used successfully in quantitative bioinformatics analysis as well.

    Finally, as stated in our MS, peptidomics investigations have been conducted for a number of different biological samples, including plasma, cerebrospinal fluid, saliva, tears, and brain tissues. To our best knowledge, there are no other reports of peptidomic analysis of wound fluids. Previously published mass spectrometry based analyses of wound fluids have used classical proteomics and N-terminomics, thus investigating very different subsets of wound fluid components. These previous studies do therefore not diminish the novelty of the current study.

    Reviewer #1:

    This paper focuses on using liquid chromatography and mass-spectrometry (LC-MS) to compare peptidome of human wound fluid. In this study, uninfected healing wound fluid and infected would fluid were evaluated for potential differences that can predict wound status and infection risk. The authors concluded differences between plasma and wound fluid as well as differences between non-inflamed/non-infected wounds fluid in term of signature of LG-MS peptidome and peptide alignment maps.

    Through their analysis they found many traditional biomarkers associated with wounds such as the cytokines IL-1β, 403 IL-6, IL-8 and TNF-α; the major novel findings come from the vast number of new peptide sequences they described, that could be used as wound biomarkers or drug targets in the future. The major counterargument for their otherwise novel findings is the same group's recent publication on wound biomarkers recently published in Frontiers in Immunology, "Bioinformatic Analysis of the Wound Peptidome Reveals Potential Biomarkers and Antimicrobial Peptides".

    Regarding our recently published paper and the relation to this manuscript, please see our comments on the public evaluation summary.

    Reviewer #2:

    The authors used mass-spectrometry to analyze the peptides that are present in wounds as a result of proteolysis. The authors thoroughly investigated multiple aspects of the methods for peptidomics. The best sample preparation was determined and robustness was shown by comparing multiple injections or multiple sample preparations. Subsequently, different types of samples were tested, i.e. normal plasma, sterile acute wound fluid and infected wound fluid, in order to be able to distinguish e.g. common proteins. Wound fluids were shown to contain more and smaller peptides than plasma. Further analysis showed clear differences in peptide profiles between wound fluids and plasma. In high inflammatory samples, which contain high levels of cytokines, the protein degradation correlated with enzymatic activity (zymograms). Many proteins were identified that were found exclusively in the low or in the high inflammation group. This will help elucidating the pathways during wound healing and/or infection but also for diagnosis or biomarker discovery.

    The conclusions of this paper are well supported by data. Although interesting differences were found between low and high inflammation, only a limited number of patient samples have been analyzed.

    Reviewer #3:

    Van der Plas et al established a mass-spectrometry based work flow for the analysis of peptidome in wound fluids. They found that wound fluids contained a higher degree of peptides as compared to plasma which is expected because of proteolytic events in wound fluids. Authors identified unique peptide patterns in healing and non-healing (infected) wounds and nicely discuss many of the identified peptides/peptide patterns and their likely roles in innate immunity, healing etc. The established methodology seems to be robust and yields interesting insights into proteolytically generated peptides in wound fluids. Authors speculate that assessing the peptidome of wounds would result in the identification of potential biomarkers for wound healing and infection.

    The manuscript is the first that determines the peptidome in wound fluids using an unbiased technology. However, the results gained are largely confirmative or "as expected" because others have previously reported an increase in peptides number in wound fluids due to proteolytic activity. Also the same group recently published a related paper without discussing it. The main novelty of the manuscript is thus more of technological interest, as long as the translational perspective (diagnostic approach) has not been demonstrated.

    Indeed, increased peptide numbers in a proteolytic environment, such as wound fluids, are to be expected and have been reported earlier. However, that does not make our results largely confirmative, as the aim of this study was not to investigate quantitative differences in peptide numbers, but to study qualitative differences in peptide patterns. Previously published mass spectrometry based analyses of wound fluids have used classical proteomics or N-terminomics, thus investigating very different subsets of wound fluid components. Therefore, these previous studies do not affect the novelty of the current study.

    Regarding our recently published paper and the relation to this manuscript, please see our comments on the public evaluation summary.

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  2. Reviewer #3 (Public Review):

    Van der Plas et al established a mass-spectrometry based work flow for the analysis of peptidome in wound fluids. They found that wound fluids contained a higher degree of peptides as compared to plasma which is expected because of proteolytic events in wound fluids. Authors identified unique peptide patterns in healing and non-healing (infected) wounds and nicely discuss many of the identified peptides/peptide patterns and their likely roles in innate immunity, healing etc. The established methodology seems to be robust and yields interesting insights into proteolytically generated peptides in wound fluids. Authors speculate that assessing the peptidome of wounds would result in the identification of potential biomarkers for wound healing and infection.

    The manuscript is the first that determines the peptidome in wound fluids using an unbiased technology. However, the results gained are largely confirmative or "as expected" because others have previously reported an increase in peptides number in wound fluids due to proteolytic activity. Also the same group recently published a related paper without discussing it. The main novelty of the manuscript is thus more of technological interest, as long as the translational perspective (diagnostic approach) has not been demonstrated.

    Read the original source
    Was this evaluation helpful?
  3. Reviewer #2 (Public Review):

    The authors used mass-spectrometry to analyze the peptides that are present in wounds as a result of proteolysis. The authors thoroughly investigated multiple aspects of the methods for peptidomics. The best sample preparation was determined and robustness was shown by comparing multiple injections or multiple sample preparations. Subsequently, different types of samples were tested, i.e. normal plasma, sterile acute wound fluid and infected wound fluid, in order to be able to distinguish e.g. common proteins. Wound fluids were shown to contain more and smaller peptides than plasma. Further analysis showed clear differences in peptide profiles between wound fluids and plasma. In high inflammatory samples, which contain high levels of cytokines, the protein degradation correlated with enzymatic activity (zymograms). Many proteins were identified that were found exclusively in the low or in the high inflammation group. This will help elucidating the pathways during wound healing and/or infection but also for diagnosis or biomarker discovery.

    The conclusions of this paper are well supported by data. Although interesting differences were found between low and high inflammation, only a limited number of patient samples have been analyzed.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #1 (Public Review):

    This paper focuses on using liquid chromatography and mass-spectrometry (LC-MS) to compare peptidome of human wound fluid. In this study, uninfected healing wound fluid and infected would fluid were evaluated for potential differences that can predict wound status and infection risk. The authors concluded differences between plasma and wound fluid as well as differences between non-inflamed/non-infected wounds fluid in term of signature of LG-MS peptidome and peptide alignment maps.

    Through their analysis they found many traditional biomarkers associated with wounds such as the cytokines IL-1β, 403 IL-6, IL-8 and TNF-α; the major novel findings come from the vast number of new peptide sequences they described, that could be used as wound biomarkers or drug targets in the future. The major counterargument for their otherwise novel findings is the same group's recent publication on wound biomarkers recently published in Frontiers in Immunology, "Bioinformatic Analysis of the Wound Peptidome Reveals Potential Biomarkers and Antimicrobial Peptides".

    Read the original source
    Was this evaluation helpful?
  5. Evaluation Summary:

    This well-done study establishes a work flow for the analysis of the peptidome of wound fluids. By doing so it enables the identification of peptide patterns associated with wounds that are healing versus non-healing. The method may therefore help to define candidate biomarkers for wound healing. Overall enthusiasm was somewhat dampened by findings previously reported by the same group and also by others.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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