Characterization of Diagenetiforms in an Expanded Proteome of the Extinct Moa (Dinornithidae): Identifying Biological, Diagenetic, Experimental Artifact, and Mislabeled Modifications in Degraded Tissues

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article is excellent and I think as someone who is not too familiar with these techniques it is an interesting and clarifying study. I appreciate the thorough review of the methods and results and the investigation into caveats such as artifacts from experiments and the laboratory environment.  The increase in knowledge of the proteome for the moa is important in itself, but also shows that the advances in extraction technologies and bioinformatics methods can all for other paleo-proteomes to be further explored for more solid data than was previously believed.  I think this paper is an important step in understanding the limitations of ancient tissue data and the reasons behind it.  Definitely an important contribution!

 

I found one potential typo on line 333, Figure 6 caption, did you want to put the word 'a' between to and single?  

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments 

The study of ancient proteins – paleoproteomics - is a quickly growing field, involving several research areas, such as molecular biology, paleontology, anthropology, and archaeology. This approach is based on the longevity and diversity of proteins to explore fundamental questions about the past. Technological advances in the last two decades have allowed increasing opportunities to better understand preservation, degradation, and recovery of the abundant bio-archive of proteins found in archaeological and paleontological records, turning it today in an expanding field. Yet, improvements of protocols and methodologies are still needed to optimize the extracted information of various biological tissues, recovered from different depositional environments. It is under this scope that the presented manuscript is a relevant contribution, allowing the author to detect not previous described PTMs for the analysed taxa besides additional modifications (see table 2, and lines 195 and 318), through a methodological improvement. All types of PTMs are clearly described and probable/possible interpretation provided.

The manuscript is well written and supported by data presented along the main text, as well, in the supplementary material.

In the discussion/conclusion sections, is it possible to explore more the influence of depositional environment and taphonomic factors in the protein degradation of ancient tissues and how this can influence the adopted/proposed methodology/procedure? Can the proposed approach be used in other biological tissues?

 Minor aspects:

-Correct spelling error in line 35: diagenetic

In sum, this manuscript is a valid contribution in the development of adequate protocols/methodologies in paleoproteomics studies, and it is worth publishing with minor revisions.

I hope my comments are useful for the author and can contribute to the improvement of their manuscript.

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents an in-depth review of the post-translational modifications undergone by the proteins present in a fossil bone from Moa, as well as diagenetic modifications. This is crucial information for many proteomics studies of fossil bone remains. Proteomics applied to fossils is a field in broad development that must be established on solid foundations. If the existence of some of these modifications has been known before, many of those described were not. In my opinion, this is a work of great scientific quality and broad applicability in the future. I congratulate the author for this interesting and necessary research.

I have only a few questions/observations:

1. Are the observed modifications found in specific areas of the molecules (I am thinking for example of collagens, which are particularly long molecules)?

2. This study was carried out on a subfossil bird bone (Moa) and it would be interesting to comment on the possibility of observing similar results on other vertebrates.

3. I think the specific Latin name of the bird under study should be included.

4. Page 9, line 241: where it says glycine, shouldn't it say glutamine?

Author Response

Please see the attached response.

Author Response File: Author Response.pdf