PONE-D-21-32984Modular automated bottom-up proteomic sample preparation for high-throughput applicationsPLOS ONE

Dear Dr. Petzold,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Partly

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

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4. If the manuscript contains new data, have the authors made this data 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: No

Reviewer #2: No

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5. Is the article 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 highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

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6. 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: The authors present an optimized workflow to prepare samples for bottom-up proteomics using automated liquid handling devices. The expected result is less than 15% cv of relative protein abundance.

Specific comments:

1. Data availability statement needs to be edited to reflect accurately where the data has been made available.

2. The authors suggest the method to be a widely applicable proteomics sample preparation workflow, and demonstrate the utility with gram-negative and fungal cell culture. However, many proteomics applications utilize tissue and complex biofluids (serum, lavage fluid, etc) that will likely require significant additional sample preparation steps. The authors should comment on the broader applicability of the method, or revise language to describe suitable sample types

3. The document(s) should be carefully proofread to eliminate typographical errors, specifically with respect to subscripts in chemical formulas.

Reviewer #2: The manuscript by Chen et al. describes a protocol for “automated” sample processing of fungal and bacterial cells for proteomic analysis. Specifically, the authors report 3 separate protocols that are carried out in a commonly used Beckman liquid handler - protein extraction from cells, normalization of protein content, and then trypsin digestion. There is some value in the described approach that could be of interest to a targeted audience; however, there are several issues that need to be addressed prior to publication consideration:

1) The reported protocol seems to be an extension of a previously published research article from their lab, which is within the guidelines of this type of article. However, the authors need to further detail the processing/automation that was mentioned in the first paper but also distinguish any differences. The protocol itself could be more detailed.

2) The authors state that this protocol was very similar to the previously published work but that the updated version was more flexible since it could “operated independently”. This point should be described more clearly and highlighted further in the protocol section if applicable.

3) Some points in the “expected results” don’t match up to the online protocol. For example, the online protocol instructs use of 5ul of sample for protein assay; however, in the results, authors state it uses 3ul of each sample.

4) Description and/or assessment of lysis/lysis efficiency aren’t clear. The main concern is that there might not be adequate representation of the entire proteome (e.g., membrane proteins or other specific classes) or perhaps variability in proteome composition from sample to sample. The authors in the previous paper use MRM to assess specific targets and in this reported protocol show low-coverage, spectral counting data. I understand the potential time savings and benefit of automation, but the authors should expand on limitation of the approach in this regard if applicable.

5) The original paper addresses getting the protein precipitate back in solution by performing “80 cycles of pipetting mixing at the maximum allowable aspiration and dispensing speed” but this is not addressed at all in newer protocol. Based on the extraction procedure used, this seems like a critical point that is important for subsequent processing steps.

6) There is concern of cross contamination between samples and reagents based on the deep well reagent reservoir used (there doesn’t seem to be any separation/ wells in the reservoir). Are there any washes (e.g., needle washes) in between steps?

7) More specifics should be added in the step that describes mixing on deck “with user defined times”. The total step duration to add Methanol shown is 3 minutes – is this mixing only or include other aspects of this overall step?

8) The authors state “In case of large protein pellets, mixing with a multichannel pipette may be necessary”. Is there a particular range you can provide to help the reader assess when that would be required? Related to this, while eliminating most manual pipetting, there are still some manual steps at critical points preventing this from being fully automated.

9) The authors should explain what day 1 and day 7 represent in Figure 1 (how long the organisms were left to grow before collection or days between processing?).

10) In Figure 2, the authors state UHPLC-MRM; however, they describe spectral counting as their quant approach. I believe MRM is copied over from the original paper where they used the MRM approach.

11) Although the mass spectrometry analysis is not necessarily part of the processing protocol, it was used to assess the protocol in terms of protein ID and quant (CV measurement across proteome). A short gradient was used, but the instrument employed should be able to identify more proteins than reported (especially if the protein loading amount on-column reported is correct). A more appropriate quant approach could be used (rather than spectral counting) to determine CVs across the dataset, which could address reproducibility in protein representation across the proteome if the LC-MS system is set up for precise LFQ-based quant. Measurement of the (high-abundance) top few hundred proteins with spectral counting is not really that informative.

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Reviewer #1: No

Reviewer #2: No

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Modular automated bottom-up proteomic sample preparation for high-throughput applications

PONE-D-21-32984R1

Dear Dr. Petzold,

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.

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Kind regards,

John Matthew Koomen, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the protocol been described in sufficient detail?

Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. If the manuscript contains new data, have the authors made this data 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

Reviewer #2: N/A

**********

5. Is the article 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 highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. 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: The authors' revised manuscript is acceptable as written. All comments/concerns have been addressed.

Reviewer #2: The authors addressed the comments from my original review.

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7. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No