PONE-D-24-50811

Superior normalization using total protein for western blot analysis of human adipocytes

PLOS ONE

Dear Dr. Kirsty Spalding,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

Specifically:

I regret to inform you that your manuscript does not have the scientific significance required by this journal and we must therefore reject it.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

For your information and guidance, any specific comments explaining why I have reached this decision and those received from reviewers, if available, are listed at the end of this letter.

Kind regards,

Dr. V. V. Sathibabu Uddandrao

Academic Editor

PLOS ONE

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Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The introduction effectively sets up the study's objective, emphasizing the need for improved normalization strategies in Western blot analysis. Consider briefly addressing why TP normalization has not yet become a universal standard, despite its advantages, to contextualize the significance of this research.

The methods section is comprehensive, but some procedural descriptions, such as the justification for excluding BSA in adipocyte isolation, could benefit from further elaboration. For example, explain how the adjusted protocol impacts the broader applicability of the findings.

In the protein extraction and quantification section, it might help to clarify why the BCA assay was chosen over other protein quantification methods.

The results are thorough, with clear visualizations supporting the text. However, Figure legends could provide more contextual explanations of key observations for readers unfamiliar with the technical nuances of Western blot normalization.

Some variability in TP and housekeeping proteins across patient samples is discussed, but it would be helpful to hypothesize more concretely on the biological factors contributing to these differences.

Consider summarizing the key advantages of TP normalization over individual housekeeping proteins in a concise table or schematic for clarity.

The discussion appropriately highlights the benefits of TP normalization. It would strengthen the manuscript to compare these findings more explicitly with studies employing other normalization methods in similar contexts (e.g., specific adipocyte studies).

Addressing the potential limitations of TP normalization due to tissue-specific tryptophan content is valuable. Consider discussing whether this limitation could extend to clinical applications or other experimental designs.

The supplementary figures are well-constructed, but it may be helpful to integrate key supplementary findings directly into the main figures for a more cohesive narrative.

Ensure all figure captions are self-contained, explaining abbreviations and experimental conditions in detail.

The conclusion strongly advocates for TP normalization but could also acknowledge scenarios where housekeeping proteins might still be appropriate, particularly for specific experimental designs.

Ensure consistency in terminology and abbreviations throughout the manuscript.

Revisit the statistical analysis details to ensure they are sufficiently described, particularly regarding how variability was quantified across replicates and normalization references.

The ethics statement is clear, but more details on the demographic and clinical background of the patient cohort might enhance reproducibility and relevance.

Reviewer #2: The authors in their studies clearly revealed that TP (Total Protein) normalization methods in western blotting are far superior to housekeeping (GLUT4), actin, and tubulin protein-based normalization methods, including BSA contaminations.

The methods that they suggested are more reliable and cost-effective, as they are not using any antibodies in their work and are strain-free too.

But studies were limited to only primary mature human adipocytes. No more comparisons/discussions with with other cell types of human samples.

Noise eliminations and contaminations, like with BSA proteins, were discussed exhaustively, but no data was not shown for noise elimination or contamination removal.

Reviewer #3: Westerberg et al. have produced a manuscript investigating two approaches to Western blot normalization, housekeeping protein normalization and total protein normalization, in adipocytes.

The concept of the manuscript is not particularly novel, as stain-free total protein normalization has been a common way of normalizing Western blot data for more than ten years. However, it could still be of particular interest if done rigorously and in a way where a generalizable improvement can be shown in adipocytes. In this study, there are, unfortunately, too many issues with the experimental design.

i) The study relies on commercially available antibodies, and it is of utmost significance to ensure that the antibodies used in the experiments are thoroughly validated for the type of study performed. There is no discussion regarding the validation of the antibodies used, and there is only accessible validation data for the antibody targeting FAH (#PA5-42049). Additionally, this antibody is referred to as an anti-FAA antibody even though the target referenced by the vendors is FAH. There is no common gene abbreviation called FAA. This antibody is also disregarded due to signal saturation, which could easily be avoided by protocol optimization. A few recommended readings on antibody validation are:

- “Antibody validation” by Bordeaux et al. from 2010, BioTechniques.

- “Enhanced validation of antibodies for research applications” by Edfors et al. from 2018, Nature Communications.

- “Antibodypedia, a Portal for Sharing Antibody and Antigen Validation Data” by Björling and Uhlén from 2008, Molecular & Cellular Proteomics.

ii) The authors only investigate the normalization in the context of one protein target. The lack of different protein targets subjected to normalization nullifies any generalization of the findings, and making any generalized claims without investigating at least a smaller data set of ten different target proteins would be ill-advised.

iii) The manuscript does not have a proper statistical evaluation that supports the claim of improved accuracy with total protein normalization, and there are no established reference values for the samples that can indicate if the observed intensities actually reflect the truth.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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For journal use only: PONEDEC3

Dear Dr. Spalding,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Jérôme Robert, PhD

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: I have gone through with the revised manuscript and found it suitable for the publication. Authors have addressed all comments

Reviewer #3: The authors seem to have misunderstood the comment regarding assessing normalization in multiple target proteins. In the study, normalization is only evaluated in the context of GLUT4. To claim that the normalization is superior for experiments conducted in primary human adipocytes, the normalization strategies must be assessed for multiple proteins of variable expression between individuals. Without this comparison, the claims of superior normalization can only be applied to GLUT4 specifically.

Additionally, there is a significant need to discuss the validation of the antibodies used beyond “all commercially validated” when using these as the basis for comparing different normalization strategies. As stated in the previous reviewer comments, there is only publicly available validation data for one of the antibodies, and a discussion and justification for the choice of these antibodies should be included in the manuscript.

As the authors state in their response, they have done a robust statistical assessment of the measurement stability. Still, stability is not the same thing as accuracy. Without any established reference values for GLUT4 in the different samples from individuals (preferably with a different methodology), there is no evidence of improved accuracy in the measurements. Accuracy is a measurement of how well the quantification reflects the actual amount of the analyte. Accuracy can, for example, be assessed by measuring the samples with an orthogonal method or by spiking a sample with different amounts of a recombinant protein. This is of significant importance when working with normalization strategies, as the normalization directly impacts the measurement values obtained from an analysis.

Reviewer #4: The manuscript entitled “Superior normalization using total protein for western blot analysis of human

adipocytes” demonstrates that total protein might be a better normalization element in WB quantification of protein lysates when studying adipose tissue and adipocytes. In general, the experiments are well designed and presented. I have few questions and comments that may improve the readability of the manuscript:

• Lines 246-253 “Overexposure of the blot showed that the expected GLUT4 signal (~54 kDa) was clearly present in the BSA-free samples while the BSA-isolated samples showed non-specific bands at ~60 kDa (S2B Fig). These results can be explained by anti-GLUT4 antibody binding non-specifically to BSA (65 kDa) leading to false positives in BSA-isolated samples but not in BSA-free samples. Thus, subsequent experiments within this study used BSA-free samples, accentuating the importance of limiting BSA, or other protein contaminants, in western blotting”. Is there a reason why the authors speculate that is the anti-GLUT4 that is cross-reacting with BSA and not the secondary antibody?

• I am curious if is there a technical/biological reason for GLUT4 selection as target gene? Notoriously, WB of membrane proteins are less clean and often characterized by double bands not very sharped associated to the glycosylated and non-glycosylated forms.

• Figure 2 shows technical replicates from OBNI individuals. Do I understand correctly that each dot represent different runs from the same lysate sample? If so, please add/reiterate this when you describe the results and in the figure legend.

• I do not see a callout for table 1.

• Sometimes μg was reported as ug (e.g., line 263, line 309). Please correct.

• Sometimes numbers were reported with comma (e.g., 2,5 – line 300), sometimes with dot (e.g., 27.3, line 320). I suggest using everywhere the dot format.

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Reviewer #1: Yes:  Umesh Kumar

Reviewer #3: No

Reviewer #4: No

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Superior normalization using total protein for western blot analysis of human adipocytes

PONE-D-24-50811R2

Dear Dr. Spalding,

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Kind regards,

Jérôme Robert, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: