Metabolomics and lipidomics in C. elegans using a single sample preparation

  1. Marte Molenaars
  2. Bauke V. Schomakers
  3. Hyung L. Elfrink
  4. Arwen W. Gao
  5. Martin A.T. Vervaart
  6. Mia L. Pras-Raves
  7. Angela C. Luyf
  8. Reuben L. Smith
  9. Mark G. Sterken
  10. Jan E. Kammenga
  11. Antoine H. C. van Kampen
  12. Georges E. Janssens
  13. Frédéric M. Vaz
  14. Michel van Weeghel
  15. Riekelt H. Houtkooper

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Abstract

Comprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand, for instance in research related to nutrition and aging. The nematode C. elegans is a key model organism in these fields, due to the large repository of available C. elegans mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans , using a single sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for instance, glycolysis and the TCA cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample.

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  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Molenaars et al., describe a protocol to extract and quantify a wide range of polar and apolar metabolites from the same C. elegans sample using methanol-chloroform based phase separation. The authors assess the method across different input amounts, in comparison to a 1-phase extraction method and through metabolic perturbations using RNAi against several metabolic enzymes. Finally, they provide a metabolomics analysis of metabolite variation across several C. elegans strains. The data are of overall high quality and presented in a clearly written manuscript.

    We really appreciate the positive words from the reviewer.

    To help assessing the value of the method to other approaches, several controls are suggested below:

    1.Fig.1: Metabolite abundance in the polar phase should be compared to 1-phase extraction methods (analogous to Fig. 2I, which compares metabolites in the apolar phase to 1-phase extraction)

    We acknowledge the apparent asymmetry in the text; comparing our two-phase method to a single phase lipidomics method indeed suggests a similar comparison for metabolomics. However, our established polar metabolomics method has always been based on this exact two-phase extraction. The current method exclusively asks whether it is possible to integrate our dedicated lipidomics platform into our established two-phase polar metabolomics method, by utilizing the apolar phase that is usually discarded. This way, the method enables comprehensive metabolomics/lipidomics screening while limiting the need of culturing twice the amount of material.

    Our manuscript does not necessarily ask the more fundamental question of the advantages of a one-phase vs two-phase extraction for polar metabolites. Interestingly, the one-phase vs two-phase metabolomics methods have been compared previously and the authors show here that the two-phase method achieved broader metabolite coverage, satisfactory extraction reproducibility, acceptable recovery and safety (DOI: 10.1038/srep38885). This is most probably due to the cHILIC column being sensitive for contamination and therefore excluding lipids from your samples is beneficial for measuring polar metabolites. We hence believe that developing a single phase polar method would appear superfluous for the purpose of this study.

    2.Are polar metabolites also detected in the apolar phase? Can the less hydrophobic lipids missing from the apolar phase detected in the polar phase?

    This is an interesting question that mostly relates to the lyso-lipids that are not detected in the lipid phase of our two-phase extraction. The first point to make is that sample solvents that are used at the final stage of extraction are not compatible between methods. In other words, the solvent we normally use for the lipids phase (xxx) cannot be injected on the cHILIC column. So, in a practical sense, we would not be able to measure these compounds, even if they would technically be dissolved in the other layer. However, we tried a few different alternative approaches to get more information on this point:

    We have attempted to integrate the lyso-lipids in the cHILIC measurements, in the polar layer, using the polar sample solvents. This was unsuccessful; no reproducible peaks, not even the internal standards, were measured. We will include a note on these results in our manuscript. We have, albeit for a different sample matrix, attempted to dissolve both layers of the two-phase extraction in the cHILIC sample solvents. While we cannot guarantee this for all metabolites, it appears that most polar metabolites are exclusively found in the polar layer. We were not able to integrate even a single peak from any of the sugar, amino acids, nucleotides, etc in the apolar layer dissolved in polar solvents. We have reconstituted both the polar and apolar layer of our two-phase extraction in 50:50 methanol:chloroform and analyzed them on the lipidomics platform. We did find some of the lipid internal standards partition to the polar phase, especially LPG (and to a lesser extent LPE and LPA) compared to for instance PE, SM, PG and PC that all end up in the apolar phase. We will include these data in the revised manuscript as a supplemental figure as it demonstrates that the lyso-lipids are poorly measured in the two-phase extraction. This is also why in the text we advise to use the dedicated one-phase extraction when interested primarily in these species.

    3.Fig.3l-n: The authors claim that extracting metabolites from the polar and apolar phases of the same sample leads to better cross-correlation than if metabolites are extracted from different samples using methods optimized for the respective metabolite classes. To provide experimental evidence, metabolite abundance should be compared directly when metabolites are extracted from the same or from different samples using suitable methods.

    We agree with this point. We will amend the text to not overstate these advantages.

    Reviewer #1 (Significance (Required)):

    The methodological and conceptual advancement of the present study is rather incremental. The authors essentially use the classical chloroform/methanol/water phase separation protocols developed by Bligh & Dyer and Folch, which have been used extensively for lipid extraction for many decades now. However, the effort to carefully measure the metabolites contained in the aqueous phase is laudable. For method validation, the authors use well-understood perturbations that yield predictable results. Overall, I consider the study more appropriate for a publication as a methods protocol, which could be of interest to the metabolomics community, rather than as a research paper.

    We agree; our goal was indeed to create and share a method, we will make sure to emphasize this in our cover letter.

    While the extraction method we use is not novel per se and based on classical extraction procedures, it is important to underscore that we are only now able to use these extractions in combination with high-resolution mass spectrometry. This opens new opportunities for basic discovery. The efficiency we achieve by using both phases of the two-phase procedure makes our method highly attractive for hypothesis generation, especially in sample sets where limited amounts of material are available.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The authors provide a detailed description of a method to analyse both polar as well as lipophilic metabolites from the same nematode sample. This provides significant advantages over methods using individual samples. Moreover and by using internal standards they establish an extremely good correlation of individual metabolites. This paper is of immediate importance for the worms community and beyond.

    We are very grateful to receive this positive response from the reviewer and for highlighting the advantages of our described method also beyond the worm community.

    **Major comments:**

    none

    **Minor comments:**

    The correction process using internal standards could be described a bit more detailed.

    In our revised manuscript, we will describe the internal standard use and corrections in more detail in the text. In summary: internal standards are selected for specific metabolites based on their Pearson correlation and %CV. Subsequently, metabolite peak areas were divided by the area of the appropriate internal standard. This corrects for any loss of sample during sample prep, for instance during the isolation of the two layers.

    Jenni Watts has written a nice Worm Book chapter on lipids which may be cited in addition to reference 17, since it covers many of the metabolites and related enzymes contained in this manuscript

    We will include a reference to this Worm book chapter reviewing fat regulation in C. elegans in our paper, thank you for the suggestion.

    Reviewer #2 (Significance (Required)):

    see above

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    The manuscript is well written and consider. However, there is room for further improvements:

    We thank the reviewer for the positive response and for the suggestions raised.

    1. Author need to write exactly how many metabolites not just >, semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, for example they did with other papers in Table 1

    We understand that this might appear vague. The notation was a compromise, based on the following considerations:

    1. The maximum number of reported metabolites can be different to the number of analyzed metabolites in a specific experiment or even a specific sample. For instance, our method is perfectly capable of measuring creatine metabolism –we have standards for these metabolites and they can be reliably measured–, however we have not yet been able to detect these metabolites in * elegans*. Some mutants also lose abundance of a certain metabolite to the point of it not being reliably measurable, which means they are filtered out in the bioinformatics.
    2. Since the initial draft of our manuscript we have been able, and will continue to be able, to add new metabolites to our analysis, as we perform a full scan over the range of m/z 50-1200. Because of this, we felt it more accurate to state that we can measure >100 metabolites, instead of a specific number.
    1. Authors also need to clarify on number of samples in the result section while describing the statistical analysis.

    We understand this point raised by the reviewer and will specify not only the number of samples, but also that they are indeed biological replicates. This will be included in the figure legends.

    Reviewer #3 (Significance (Required)):

    This might be interesting paper for the research community who work with C.elegans (metabolism or in general)

    Thank you, we are in fact utilizing this double extraction for other non-worm samples such as mice an human tissues and we believe this could also benefit the research community beyond the model organism C. elegans.

    The authors must deposit the raw data and make it available for the public, so they could also benefit from this good work.

    It is our full intention to share our data in a convenient and standardized way through for instance the MetaboLights database (https://www.ebi.ac.uk/metabolights/). We agree and changes will be implemented as suggested.

    Reviewer #4 (Evidence, reproducibility and clarity (Required)):

    **Summary:** The authors present a method for extraction of both lipid and polar metabolites from the model organism C. elegans. This extraction method is based on the well-established Blyth and Dyer method, with a slight modification to retain and utilize both the organic and non-polar fractions for LCMS analysis. They applied and tested this method against a monophasic extraction utilizing the same solvent system. They report that there is a loss of metabolites in the non-polar fraction to the polar fraction (of more polar metabolites) and small differences between the monophasic and biphasic extractions. They also expanded on the linearity of the extraction efficiency by increasing the number of worms. Further they applied the single extraction method to both knockdown mutants of C. elegans and Recombinant Inbred Lines derived from N2 and the natural isolate CB4856 to determine whether this method would still be able to differentiate the metabolome between the genetically different C. elegans populations.

    We thank the reviewer for their comments and suggestions.

    **Major comments:**

    *Are the key conclusions convincing?*

    As a whole the conclusions are convincing and valid.

    We appreciate that the reviewer considers our work convincing and valid.

    *Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?*

    The use of the adjective "robust" is, to an extent, erroneous. As defined, a robust method implies that the method is capable of withstanding small (deliberate or not) changes or variations. In this case the robustness of the method was not assessed and not clear how replication was carried out.

    We have in fact performed analysis on both biological replicates and repeated injections of pooled samples to determine robustness. We will clarify the biological replicates in the text and will place the pooled QC samples in the main text with additional explanation and relevant statistics such as % coefficient of variance (%CV) between them. For clarity, we plotted %CV of all polar as well as apolar metabolites. For polar metabolites 97% of the metabolites had a %CV lower than 30. For apolar metabolites 86% of the metabolites had a %CV lower than 30.

    *Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.*

    Reproducibility would need to be assessed/quantified to establish how robust the method is. Even though linearity with an increase in the number of worms is a good indication, it does not satisfactorily establish the robustness of the method. The use of replicates to assess the agreement between measurements (i.e. bland-Altman plots), linearity as well as coefficients of variation (included in the sup material but not clear in the body of the manuscript) would characterize the methods best. The isolation of each variance originating from instrumental (pooled quality controls), biological (biological replication) and sample preparation (multiple extractions from the same biological source) is critical.

    We have these data and will elaborate on this in our revised manuscript. We will discuss the quality control samples more prominently in the main body of the manuscript, and show one or more figures that specifically address both analytical and biological variance (see rebuttal figure 2). In summary, we assessed this variance using (a) a repeated injection of a pooled QC sample, and (b) biological replicates prepared individually. Especially the latter condition, in which we assess biological variance is representative for the actual method application. The %CV under these conditions is ≤20% for the majority of metabolites, which is why we consider our method robust.

    *Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.*

    The suggested experiments are in-fact just further analysis with the already collected data. There would be no need for further experiments, however it is not clear whether pooled QCs/or reference materials were used and the number of replicates per experimental design.

    All the data are available. These analyses will be included in the revision.

    *Are the data and the methods presented in such a way that they can be reproduced?*

    The methods are very well described. My only comment is to address how the replicates were grown/created and how many per strain/group. If the replicate measurements were done on the same samples (repeated injections), I believe that would weaken the findings (if not invalidate them altogether), however if these were biological replicates from independent starting populations the findings are valid and convincing.

    We performed bona fide biological replicates. We will explicitly mention this in the paper together with the other descriptions of our validation protocols.

    *Are the experiments adequately replicated and statistical analysis adequate?*

    As per my above comments.

    **Minor comments:**

    *Specific experimental issues that are easily addressable.*

    It is not clear how the sample preparation process was carried out (randomization, run order, QCs etc). As per the guidelines widely accepted from –Broadhurst, D., Goodacre, R., Reinke, S.N. et al. Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies. Metabolomics 14, 72 (2018). https://doi.org/10.1007/s11306-018-1367-3.

    We will provide details on the analysis itself in a table. In summary: Samples were measured in a random order, with blanks and QC samples throughout the run.

    *Are prior studies referenced appropriately?*

    A major reference that has applied this extraction method before in the same model organism is missing:

    Castro, C., Sar, F., Shaw, W.R. et al. A metabolomic strategy defines the regulation of lipid content and global metabolism by Δ9 desaturases in Caenorhabditis elegans. BMC Genomics 13, 36 (2012). https://doi.org/10.1186/1471-2164-13-36

    We will include this paper in our references. We would like to note though that this method requires not just an LC system to analyze lipids, but also GC with additional derivatization steps. Our method achieves comprehensive lipidomics using a single technique and no additional derivatization.

    Further a recent publication that goes beyond the work described by the authors using similar approach:

    MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses. Ernesto S. Nakayasu, Carrie D. Nicora, Amy C. Sims, Kristin E. Burnum-Johnson, Young-Mo Kim, Jennifer E. Kyle, Melissa M. Matzke, Anil K. Shukla, Rosalie K. Chu, Athena A. Schepmoes, Jon M. Jacobs, Ralph S. Baric, Bobbie-Jo Webb-Robertson, Richard D. Smith, Thomas O. Metz mSystems May 2016, 1 (3) e00043-16; DOI: 10.1128/mSystems.00043-16

    We will also include this paper, reporting 51 polar metabolites and 84 lipid species, in our references. While we recognize that they also make use of both phases and the protein pellet, we think our method is much more practical in several key ways:

    Our metabolomics platform provides twice as many species and our lipids platform exceeds their analytical capabilities 10 fold. This means a far better coverage of differences within metabolite and lipid classes, allowing for far more intricate patterns to be detected. We show this for instance in our plots comparing carbon chain length to degree of saturation (Fig 4 and S2 in original manuscript); a comparison that is only possible with the data density that our method offers. The MPLEx metabolomics method also requires the use of a GC system and derivatization steps, while our method does not, making it much more user friendly and requiring only a single analytical system.

    *Are the text and figures clear and accurate?*

    Yes

    *Do you have suggestions that would help the authors improve the presentation of their data and conclusions? *

    The figures, overall are of exceptional quality.

    As per current scientific consensus, Box plots should also be overlaid with the actual datapoints (which was aptly done for the bar charts and other plots).

    The supplementary data even though comprehensive is hard to understand. A "readme" file detailing what data each file contains would improve readability and comply with FAIR principles.

    We agree that a readme file would make the supplemental data more understandable. We will provide such a file. For the box plots we will show the actual data points in our revised manuscript.

    Reviewer #4 (Significance (Required)):

    Even though the approach is not novel and has long been used in Natural Products Chemistry and in other organisms, it's highly significant to set an extraction method standard for the field of C. elegans metabolomics (including myself doing metabolomics and natural products chemistry with LCMS and NMR). However, this manuscript does not cover the technical aspects of the method with sufficient depth to hallmark this method as the standard for the field. Further information is needed to fill the missing gaps (as highlighted by the authors). Ratios between solvent and biological material amounts, reproducibility, recovery rates (even though buried in the supplementary files) and metabolite coverage are still missing.

    As a side note, the disparity between the monophasic and biphasic extractions could be overcome by a sequential extraction of the same sample, with no incurred cost on performance (and removing the much-dreaded pipetting uncertainty near the line between solvents).

    The second aspect of the manuscript, which initially was a welcoming idea (and important), became >50% of the manuscript creating a disconnect between the information set by the abstract and introduction and the results/conclusion. The work is extremely relevant in both sections of the manuscript, but the technical aspect is still lacking details and/or analysis.

    Strongly suggested: explicit compliance with the minimum reporting standards as per the Metabolomics Standards Initiative (MSI) and deposition of the data to a metabolomics repository (i.e. Metabolights or Metabolomics Workbench). These are internationally accepted requirements for metabolomics publications.

    We are aware that the extraction itself is an analytical chemistry staple. However, it is precisely in this fact that we find novelty. It should be noted that both of the other papers mentioned by the reviewers that have attempted to integrate lipidomics and metabolomics have had to resort to labor intensive (as well as possibly expensive and destructive) derivatization steps and a separate analysis on GC. Our method does not have these requirements. It is indeed a single and very common extraction, after which each dried phase is reconstituted and immediately injected. But this simplicity is not a concession, as our metabolome coverage is easily more comprehensive than the other mentioned methods. We therefore feel that this simplicity should not discount our currently presented method, but be considered an additional advantage.

    Sequential extractions may be an option to consider. However, we feel like they are less user friendly and unneeded. Because we use internal standards, it is never an issue to pipet slightly more or less of any particular sample; making it easy to avoid the line between solvents.

    We will explicitly clarify where we already comply with the standards (such as the analysis of biological replicates and repeated injection of a QC sample) and are confident we can add figures and further information such as deposition of our data to comply with the rest.

    REFEREES CROSS-COMMENTING

    Completely agree with reviewer #1 comments, they are on point and I completely missed it. Relevant and should be addressed.

    Reviewers #2 points out work worth acknowledging, the internal standard work was quite thorough and well designed.

    Reviewer #3 and my comments overlap nicely, the need for further description of samples/replication and deposition of data in a metabolomics repository.

    Further work is required to make this a good publication and standard for the field, without this extra work addressing the reviewers comments I feel this work could be to certain degree misleading and/or incomplete putting in cause its publication potential.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #4

    Evidence, reproducibility and clarity

    Summary:

    The authors present a method for extraction of both lipid and polar metabolites from the model organism C. elegans. This extraction method is based on the well-established Blyth and Dyer method, with a slight modification to retain and utilize both the organic and non-polar fractions for LCMS analysis. They applied and tested this method against a monophasic extraction utilizing the same solvent system. They report that there is a loss of metabolites in the non-polar fraction to the polar fraction (of more polar metabolites) and small differences between the monophasic and biphasic extractions. They also expanded on the linearity of the extraction efficiency by increasing the number of worms. Further they applied the single extraction method to both knockdown mutants of C. elegans and Recombinant Inbred Lines derived from N2 and the natural isolate CB4856 to determine whether this method would still be able to differentiate the metabolome between the genetically different C. elegans populations.

    Major comments:

    Are the key conclusions convincing?

    As a whole the conclusions are convincing and valid.

    Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The use of the adjective "robust" is, to an extent, erroneous. As defined, a robust method implies that the method is capable of withstanding small (deliberate or not) changes or variations. In this case the robustness of the method was not assessed and not clear how replication was carried out.

    Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Reproducibility would need to be assessed/quantified to establish how robust the method is. Even though linearity with an increase in the number of worms is a good indication, it does not satisfactorily establish the robustness of the method. The use of replicates to assess the agreement between measurements (i.e. bland-Altman plots), linearity as well as coefficients of variation (included in the sup material but not clear in the body of the manuscript) would characterize the methods best. The isolation of each variance originating from instrumental (pooled quality controls), biological (biological replication) and sample preparation (multiple extractions from the same biological source) is critical.

    Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    The suggested experiments are in-fact just further analysis with the already collected data. There would be no need for further experiments, however it is not clear whether pooled QCs/or reference materials were used and the number of replicates per experimental design.

    Are the data and the methods presented in such a way that they can be reproduced?

    The methods are very well described. My only comment is to address how the replicates were grown/created and how many per strain/group. If the replicate measurements were done on the same samples (repeated injections), I believe that would weaken the findings (if not invalidate them altogether), however if these were biological replicates from independent starting populations the findings are valid and convincing.

    Are the experiments adequately replicated and statistical analysis adequate?

    As per my above comments.

    Minor comments:

    Specific experimental issues that are easily addressable.

    It is not clear how the sample preparation process was carried out (randomization, run order, QCs etc). As per the guidelines widely accepted from -

    Broadhurst, D., Goodacre, R., Reinke, S.N. et al. Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies. Metabolomics 14, 72 (2018). https://doi.org/10.1007/s11306-018-1367-3.

    Are prior studies referenced appropriately?

    A major reference that has applied this extraction method before in the same model organism is missing:

    Castro, C., Sar, F., Shaw, W.R. et al. A metabolomic strategy defines the regulation of lipid content and global metabolism by Δ9 desaturases in Caenorhabditis elegans. BMC Genomics 13, 36 (2012). https://doi.org/10.1186/1471-2164-13-36

    Further a recent publication that goes beyond the work described by the authors using similar approach:

    MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses Ernesto S. Nakayasu, Carrie D. Nicora, Amy C. Sims, Kristin E. Burnum-Johnson, Young-Mo Kim, Jennifer E. Kyle, Melissa M. Matzke, Anil K. Shukla, Rosalie K. Chu, Athena A. Schepmoes, Jon M. Jacobs, Ralph S. Baric, Bobbie-Jo Webb-Robertson, Richard D. Smith, Thomas O. Metz mSystems May 2016, 1 (3) e00043-16; DOI: 10.1128/mSystems.00043-16

    Are the text and figures clear and accurate?

    Yes

    *Do you have suggestions that would help the authors improve the presentation of their data and conclusions? *

    The figures, overall are of exceptional quality. As per current scientific consensus, Box plots should also be overlaid with the actual datapoints (which was aptly done for the bar charts and other plots). The supplementary data even though comprehensive is hard to understand. A "readme" file detailing what data each file contains would improve readability and comply with FAIR principles.

    Significance

    Even though the approach is not novel and has long been used in Natural Products Chemistry and in other organisms, it's highly significant to set an extraction method standard for the field of C. elegans metabolomics (including myself doing metabolomics and natural products chemistry with LCMS and NMR). However, this manuscript does not cover the technical aspects of the method with sufficient depth to hallmark this method as the standard for the field. Further information is needed to fill the missing gaps (as highlighted by the authors). Ratios between solvent and biological material amounts, reproducibility, recovery rates (even though buried in the supplementary files) and metabolite coverage are still missing.

    As a side note, the disparity between the monophasic and biphasic extractions could be overcome by a sequential extraction of the same sample, with no incurred cost on performance (and removing the much-dreaded pipetting uncertainty near the line between solvents).

    The second aspect of the manuscript, which initially was a welcoming idea (and important), became >50% of the manuscript creating a disconnect between the information set by the abstract and introduction and the results/conclusion. The work is extremely relevant in both sections of the manuscript, but the technical aspect is still lacking details and/or analysis.

    Strongly suggested: explicit compliance with the minimum reporting standards as per the Metabolomics Standards Initiative (MSI) and deposition of the data to a metabolomics repository (i.e. Metabolights or Metabolomics Workbench). These are internationally accepted requirements for metabolomics publications.

    REFEREES CROSS-COMMENTING

    Completely agree with reviewer #1 comments, they are on point and I completely missed it. Relevant and should be addressed.

    Reviewers #2 points out work worth acknowledging, the internal standard work was quite thorough and well designed.

    Reviewer #3 and my comments overlap nicely, the need for further description of samples/replication and deposition of data in a metabolomics repository.

    Further work is required to make this a good publication and standard for the field, without this extra work addressing the reviewers comments I feel this work could be to certain degree misleading and/or incomplete putting in cause its publication potential.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    The manuscript is well written and consider. However, there is room for for further improvements,

    1. Author need to write exactly how many metabolites not just >, semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, for example they did with other papers in Table 1

    2)Authors also need to clarify on number of samples in the result section while describing the statistical analysis.

    Significance

    This might be interesting paper for the research community who work with C.elegans (metabolism or in general)

    The authors must deposit the raw data and make it available for the public,so they could also benefit from this good work.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The authors provide a detailed description of a method to analyse both polar as well as lipophilic metabolites from the same nematode sample. This provides significant advantages over methods using individual samples. Moreover and by using internal standards they establish an extremely good correlation of individual metabolites. This paper is of immediate importance for the worms community and beyond.

    Major comments: none

    Minor comments:

    The correction process using internal standards could be described a bit more detailed.

    Jenni Watts has written a nice Worm Book chapter on lipids which may be cited in addition to reference 17, since it covers many of the metabolites and related enzymes contained in this manuscript

    Significance

    see above

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Molenaars et al., describe a protocol to extract and quantify a wide range of polar and apolar metabolites from the same C. elegans sample using methanol-chloroform based phase separation. The authors assess the method across different input amounts, in comparison to a 1-phase extraction method and through metabolic perturbations using RNAi against several metabolic enzymes. Finally, they provide a metabolomics analysis of metabolite variation across several C. elegans strains. The data are of overall high quality and presented in a clearly written manuscript.

    To help assessing the value of the method to other approaches, several controls are suggested below:

    1.Fig.1: Metabolite abundance in the polar phase should be compared to 1-phase extraction methods (analogous to Fig. 2I, which compares metabolites in the apolar phase to 1-phase extraction)

    2.Are polar metabolites also detected in the apolar phase? Can the less hydrophobic lipids missing from the apolar phase detected in the polar phase?

    3.Fig.3l-n: The authors claim that extracting metabolites from the polar and apolar phases of the same sample leads to better cross-correlation than if metabolites are extracted from different samples using methods optimized for the respective metabolite classes. To provide experimental evidence, metabolite abundance should be compared directly when metabolites are extracted from the same or from different samples using suitable methods.

    Significance

    The methodological and conceptual advancement of the present study is rather incremental. The authors essentially use the classical chloroform/methanol/water phase separation protocols developed by Bligh & Dyer and Folch, which have been used extensively for lipid extraction for many decades now. However, the effort to carefully measure the metabolites contained in the aqueous phase is laudable. For method validation, the authors use well-understood perturbations that yield predictable results. Overall, I consider the study more appropriate for a publication as a methods protocol, which could be of interest to the metabolomics community, rather than as a research paper.

  6. Version published to 10.1101/2020.07.06.190017 on bioRxiv