From the journal RSC Chemical Biology Peer review history

17-Apr-2022

Dear Professor Zaro:

Manuscript ID: CB-ART-03-2022-000076
TITLE: Proteomic Characterization of Phagocytic Primary Human Monocyte-Derived Macrophages

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Prof Gonçalo Bernardes

Associate Editor, RSC Chemical Biology

************

Reviewer 1

In this manuscript, Volk et al use global proteomic analysis to evaluate the levels of proteins in human macrophages +/- interferon gamma. They notably identified ~6000 proteins with high-confidence, and found a number (including IDO-1) that are significantly upregulated after IFg treatment. They also used HPG treatment and enrichment to identify newly synthesized proteins after IFg treatment. Overall, the proteomic data is solid and this dataset will be useful for those studying macrophage biology. However, the study doesn’t look in depth into any biology of this dataset, and doing so would undoubtedly increase the impact of these findings.

Specific points:

1. The IDO gel in 1D does not appear to have a gel image in my version of the manuscript (computer glitch?)
2. Some of the “loading control” bands are a bit hard to see – could they choose other ones that are more clear?
3. The authors identified a number of proteins whose synthesis is increased after IFg. However, these proteins are not named or analyzed in any way. Some analysis of these proteins would be interesting and increase the impact of this manuscript.

Reviewer 2

The manuscript by Volk et al entitled “Proteomic Characterization of Phagocytic Primary Human Monocyte-Derived Macrophages” describes an efficient proteomic workflow to characterize changes in macrophages after stimulation. The approaches developed here are rigorous and will help advance the field in study macrophage activation/stimulation. In general, the experiments are well designed and the paper is very easy to read. The approach should be widely applicable to multiple macrophage stimulation experiments in mixed culture settings. I have some comments related to points of clarity that should be addressed .

1) Could the authors please comment on why the concentration if IF-g (100U/mL) and time point (72 h) were chosen? Was a concentration gradient and/or time course performed? Also, could the authors also report the concentration of IF-g in mg/mL (or molarity)?
2) For Figure 4, the authors should consider increasing the size of the macrophage to show that the alkyne probe enters the cells and is not on the surface.
3) The method section is well written. For all cell lysate preparation, could the authors comment if their preparation would allow for analysis of membrane proteins or do their methods bias towards cytoplasmic proteins?
4) The authors conclude their results section with the observation that the data show +Stim macrophages synthesize more protein than -stim macrophages. Could the authors expand on this and comment if specific proteins identified in their original screen (Fig 2) are also represented in the newly synthesized protein data set?
5) Do the authors think that the stimulant (i.e. IF-g) influences the proteins that are synthesized? If a different stimulant was used, would the results change? Perhaps this could be commented on in the discussion.

3 May 2022

Prof. Bernardes,

We are pleased to resubmit our manuscript entitled “Proteomic Characterization of Phagocytic Primary Human Monocyte-Derived Macrophages” for consideration as an article in RSC Chemical Biology. We thank you and the reviewers for a thorough evaluation of our manuscript, and we feel that the changes made in response have strengthened the rigor and presentation of our work.

In our revised manuscript we present new analyses, revised figures, additional experimental details, and an elaborated discussion. Please see the attached point-by-point rebuttal for specific reviewer comments.

Best Wishes,

Balyn W. Zaro

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters:

Referee: 1

1. The IDO gel in 1D does not appear to have a gel image in my version of the manuscript (computer glitch?)

We apologize for this technical difficulty. On our computers, we are able to see the gel. We have ensured that this is the case in our resubmission.

2. Some of the “loading control” bands are a bit hard to see – could they choose other ones that are more clear?

We have adjusted the loading control gel images to improve their contrast in Figure 2D.

3. The authors identified a number of proteins whose synthesis is increased after IFg. However, these proteins are not named or analyzed in any way. Some analysis of these proteins would be interesting and increase the impact of this manuscript.

We now include additional analysis of our synthesis (HPG) dataset and text about specific proteins. The text reads:

“Having characterized our dataset through high-level analyses, we wanted to determine if specific proteins found to be sensitive to IF-g treatment could be corroborated by previous literature reports. Proteins that were uniquely “Made in + Stim” including, Complement C2 (CO2), C1 inhibitor (IC1) and Caspase-7 (CASP7), among others, have been previously reported to be upregulated in response to IF-g39-41. Conversely, IF-g has been shown to downregulate mTOR in macrophages and this finding is recapitulated in our BONCAT studies42.”

We also cross-reference proteins that are exclusively synthesized as “Made in - Stim” or “Made in + Stim” with our whole protein analysis. The text reads:

“Finally, we cross-referenced our + Stim/- Stim and “Made in + Stim”/”Made in - Stim” datasets, focusing on proteins that were unique to either “Made in + Stim” or “Made in - Stim”. A majority of proteins detected in these data were previously identified in the whole protein analysis (348 of 378 proteins under + Stim conditions and 41 of 47 proteins under - Stim conditions). However, we were intrigued to see that very few proteins that were detectable as both + Stim and ”Made In + Stim Only” (17 proteins of 348) or - Stim and “Made in - Stim Only” (2 proteins of 41, Supplemental Figure 7A). Future investigations involve exploring the possibility of differential protein half-lives in response to macrophage stimulation, which would potentially require increased rates of synthesis to maintain comparable protein levels. These analyses highlight the value of performing both whole-protein mass spectrometry experiments and pulse-type chemical proteomics BONCAT studies over a defined time window. Gratifyingly, statistical overrepresentation analysis of biological processes in the 17 proteins overlapping as both + Stim and ”Made In + Stim Only” revealed enrichment of processes associated with innate immune response (Supplemental Figure 7B).”

Referee: 2

1. Could the authors please comment on why the concentration if IF-g (100U/mL) and time point (72 h) were chosen? Was a concentration gradient and/or time course performed? Also, could the authors also report the concentration of IF-g in mg/mL (or molarity)?

We selected this concentration and timeframe based upon previous literature reports, which are now referenced in the manuscript. A concentration gradient and time course were not conducted. IF-g treatment was performed at 5 ng/mL, and we now include these units alongside 100U/mL. Our goal with these conditions was to ensure that we could observe an increase in phagocytic capacity between treatment and no treatment.

2. For Figure 4, the authors should consider increasing the size of the macrophage to show that the alkyne probe enters the cells and is not on the surface.

We have adjusted the figure accordingly.

3. The method section is well written. For all cell lysate preparation, could the authors comment if their preparation would allow for analysis of membrane proteins or do their methods bias towards cytoplasmic proteins?

Our lysis methods allow for detection of both membrane and soluble proteins. We have included this detail in our methods section where relevant.

4. The authors conclude their results section with the observation that the data show +Stim macrophages synthesize more protein than -stim macrophages. Could the authors expand on this and comment if specific proteins identified in their original screen (Fig 2) are also represented in the newly synthesized protein data set?

We now include additional analysis to compare proteins that are synthesized +/- Stim and their steady-state relative abundance. Data is reported in Supplemental Figure 7. For each of cross-reference for specific proteins, we also now include the whole protein +Stim/-Stim ratio in the HPG data tables (Tables 8-11). The main text now reads:

“Finally, we cross-referenced our + Stim/- Stim and “Made in + Stim”/”Made in - Stim” datasets, focusing on proteins that were unique to either “Made in + Stim” or “Made in - Stim”. A majority of proteins detected in these data were previously identified in the whole protein analysis (348 of 378 proteins under + Stim conditions and 41 of 47 proteins under - Stim conditions). However, we were intrigued to see that very few proteins that were detectable as both + Stim and ”Made In + Stim Only” (17 proteins of 348) or - Stim and “Made in - Stim Only” (2 proteins of 41, Supplemental Figure 7A). Future investigations involve exploring the possibility of differential protein half-lives in response to macrophage stimulation, which would potentially require increased rates of synthesis to maintain comparable protein levels. These analyses highlight the value of performing both whole-protein mass spectrometry experiments and pulse-type chemical proteomics BONCAT studies over a defined time window. Gratifyingly, statistical over-representation analysis of biological processes in the 17 proteins overlapping as both + Stim and ”Made In + Stim Only” revealed enrichment of processes associated with innate immune response (Supplemental Figure 7B).”

5. Do the authors think that the stimulant (i.e. IF-g) influences the proteins that are synthesized? If a different stimulant was used, would the results change? Perhaps this could be commented on in the discussion.

We hypothesize that different stimulants likely influence the repertoire of proteins synthesized. Commentary related to this hypothesis is included in our discussion. The end of our discussion now reads:

“Due to non-specific targeting by the innate immune system, we anticipate inflammatory stimuli from mammalian or pathogenic origins may result in a similar proteomic signature. This may contrast with anti-inflammatory stimuli which could downregulate proteins identified in this report. In future work we plan to explore a diverse panel of stimuli to identify proteins which may be attractive broad-spectrum therapeutic targets. Additionally, protein expression signatures for a particular macrophage phenotype (for example, pro- or anti-inflammatory) could also serve an experimental readout in future phenotypic screens for innate immunomodulatory molecules.”

12-May-2022

Dear Professor Zaro:

Manuscript ID: CB-ART-03-2022-000076.R1
TITLE: Proteomic Characterization of Phagocytic Primary Human Monocyte-Derived Macrophages

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Prof Gonçalo Bernardes

Associate Editor, RSC Chemical Biology

Reviewer 1

Looks fine to me.