Proteomic method to extract, concentrate, digest and enrich peptides from fossils with coloured (humic) substances for mass spectrometry analyses

Humic substances are breakdown products of decaying organic matter that co-extract with proteins from fossils. These substances are difficult to separate from proteins in solution and interfere with analyses of fossil proteomes. We introduce a method combining multiple recent advances in extraction protocols to both concentrate proteins from fossil specimens with high humic content and remove humics, producing clean samples easily analysed by mass spectrometry (MS). This method includes: (i) a non-demineralizing extraction buffer that eliminates protein loss during the demineralization step in routine methods; (ii) filter-aided sample preparation (FASP) of peptides, which concentrates and digests extracts in one filter, allowing the separation of large humics after digestion; (iii) centrifugal stage tipping, which further clarifies and concentrates samples in a uniform process performed simultaneously on multiple samples. We apply this method to a moa fossil (approx. 800–1000 years) dark with humic content, generating colourless samples and enabling the detection of more proteins with greater sequence coverage than previous MS analyses on this same specimen. This workflow allows analyses of low-abundance proteins in fossils containing humics and thus may widen the range of extinct organisms and regions of their proteomes we can explore with MS.


Recommendation?
Major revision is needed (please make suggestions in comments) Comments to the Author(s) Schroeter et al. build in the current manuscript on their earlier works in an effort to mitigate a potentially major issue in palaeoproteomics -the presence of large amounts of humics in fossil bone interfering during protein extraction and MS analysis. This is a relevant question to resolve, as is indicated by the recent literature on this topic as well.
The major issue with the manuscript is that an internal control is lacking. That is, claims of relevance made in the paper of enhanced protein extraction through either Method 1 and Method 2 refer to extractions and data presented in a previous paper (Cleland et al., 2016, PRSB; Cleland et al., 2012, Plos One). However, recent research in protein preservation has shown remarkable variation of within-bone protein preservation, necessitating that extraction comparisons are performed on homogenized bone powders. Further issues with comparing the enhanced results derive from an absence in protein or peptide quantification, differences in MS set-up, and column chemistry. Therefore, the main conclusion of the paper could be seen as suggestive.
Minor comments: -Lines 54-65. This is one massive sentence. Please break up in smaller sentences.
-Lines 85-98. The four elements of the used extraction methods are presented as novel. However, each of these has already appeared in the palaeoproteomic literature at least once. To avoid the suggestion that all four elements are introduced here for the first time, references to relevant literature are justified.
-Line 96: The re-occurring claim that this bone specimen has a particularly high humic content requires quantification. -Line 126 vs line 147: You mention "a small piece", but later refer to approx. 250 mg., which is a large piece of bone, especially of this age. These two statements are in conflict, especially as "small" frequently refers to samples under 20mg in the isotope, collagen, and bone proteome literature.
-Line 257: I think you mean smaller than 3 kDa, not larger than 3 kDa.
In addition to addressing all of the reviewers' and editor's comments please also ensure that your revised manuscript contains the following sections as appropriate before the reference list: • Ethics statement (if applicable) If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data have been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that have been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-181433 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Kind  Comments to the Author(s) Schroeter et al. build in the current manuscript on their earlier works in an effort to mitigate a potentially major issue in palaeoproteomics -the presence of large amounts of humics in fossil bone interfering during protein extraction and MS analysis. This is a relevant question to resolve, as is indicated by the recent literature on this topic as well.
The major issue with the manuscript is that an internal control is lacking. That is, claims of relevance made in the paper of enhanced protein extraction through either Method 1 and Method 2 refer to extractions and data presented in a previous paper (Cleland et al., 2016, PRSB; Cleland et al., 2012, Plos One). However, recent research in protein preservation has shown remarkable variation of within-bone protein preservation, necessitating that extraction comparisons are performed on homogenized bone powders. Further issues with comparing the enhanced results derive from an absence in protein or peptide quantification, differences in MS set-up, and column chemistry. Therefore, the main conclusion of the paper could be seen as suggestive.
Minor comments: -Lines 54-65. This is one massive sentence. Please break up in smaller sentences. -Lines 85-98. The four elements of the used extraction methods are presented as novel. However, each of these has already appeared in the palaeoproteomic literature at least once. To avoid the suggestion that all four elements are introduced here for the first time, references to relevant literature are justified. -Line 96: The re-occurring claim that this bone specimen has a particularly high humic content requires quantification. -Line 126 vs line 147: You mention "a small piece", but later refer to approx. 250 mg., which is a large piece of bone, especially of this age. These two statements are in conflict, especially as "small" frequently refers to samples under 20mg in the isotope, collagen, and bone proteome literature.
-Line 257: I think you mean smaller than 3 kDa, not larger than 3 kDa.

Reviewer: 2
Comments to the Author(s) It is suggested that the methods section of the article should be revised to improve its clarity.

Do you have any ethical concerns with this paper? Yes
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The additional supplemental table does not address the reviewer's previously expressed concerns.

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes I do not wish to add any more comments as it would not be fair on the authors, but I would strongly suggest to change the title as follows "A proteomic method to extract, concentrate, digest and enrich peptides from from sub-fossil bones with colored (humic) substances for mass spectrometry analyses". This is because: a) the paper only deals with bone, which has different characteristics than other substrates for paleoproteomics (for example skipping demineralization is not appropriate in all cases); b) "fossil" is a difficult word and often implies no organics and old age -using "sub-fossil" is useful to indicate that you do have organics; c) "abundant" still implies quantitation, which is not available in the paper; d) technically the authors do not provide chemical evidence that the colored substances are in fact "humics" -better to focus on the color aspect.

12-Mar-2019
Dear Dr Schroeter: Manuscript ID RSOS-181433.R1 entitled "A proteomic method to extract, concentrate, digest, and enrich peptides from fossils with abundant humics for mass spectrometry analyses" which you submitted to Royal Society Open Science, has been reviewed. The comments of the reviewer(s) are included at the bottom of this letter.
Please submit a copy of your revised paper before 04-Apr-2019. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available we may invite new reviewers.
To revise your manuscript, log into http://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision. Revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you must respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". Please use this to document how you have responded to the comments, and the adjustments you have made. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response.
In addition to addressing all of the reviewers' and editor's comments please also ensure that your revised manuscript contains the following sections before the reference list: • Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data have been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that have been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
• Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch.
Kind regards, Andrew Dunn Royal Society Open Science Editorial Office Royal Society Open Science openscience@royalsociety.org on behalf of Prof Kevin Padian (Subject Editor) openscience@royalsociety.org Editorial office comments: Unfortunately, in an earlier round of review, the comments supplied by a reviewer that were intended for the author as well as the Editor were not communicated to the authors, as the comments had been included in the comments to the Editor. Following our standard processes, these comments were not forwarded to the authors (comments supplied in confidence to the Editor are not generally communicated to the authors); however, from the comments of the reviewer, it would seem these comments were intended for the author as well. With this in mind, we now supply the substantive elements of these comments immediately below, with the current round of comments provided below this. We apologise for this state of affairs, and hope you'll address the comments in this round of review. ==Original comments== there are inconsistencies between different statements in the materials and methods section. For example, on page 6 it is stated that the bone sample was divided into 10 aliquots of which 5 were treated with NaOH and then all 10 samples were treated with the 400/200/4 solution. But on the following page (p7) it is stated that there were three sample sets, one of which had NaOH but no 400/200/4 treatment, which is inconsistent with the description given on the preceding page. Figure 1 is of little help in undestanding the experimental protocol. Overall, this article if revised could make a useful contribution to the palaeoproteomics literature but as submitted I think that it fails to meet the journal's requirement that published work should be "scientifically sound, in which the methodology is rigorous and the conclusions fully supported by the data".
Reviewer comments to Author: Reviewer: 3 Comments to the Author(s) I have reviewed the revisions made by the authors in order to address the first round of reviews. The authors have responded in an adequate way to all queries. I do not wish to add any more comments as it would not be fair on the authors, but I would strongly suggest to change the title as follows "A proteomic method to extract, concentrate, digest and enrich peptides from from sub-fossil bones with colored (humic) substances for mass spectrometry analyses". This is because: a) the paper only deals with bone, which has different characteristics than other substrates for paleoproteomics (for example skipping demineralization is not appropriate in all cases); b) "fossil" is a difficult word and often implies no organics and old age -using "sub-fossil" is useful to indicate that you do have organics; c) "abundant" still implies quantitation, which is not available in the paper; d) technically the authors do not provide chemical evidence that the colored substances are in fact "humics" -better to focus on the color aspect.

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
In the manuscript entitled "A proteomic method to extract, concentrate, digest, and enrich peptides from fossils with colored (humic) substances for mass spectrometry analyses", Schroeter and co-authors describe an protocol to extract ancient protein residues from bone samples highly infiltrated with coloured (humic) substances. As the authors point out, the challenge the method aims at addressing is significant. The study clearly describes how the method presented clearly leads to higher recoveries in terms of proteins peptides and PSMs. In the current version the manuscript is clearly readable, the experimental protocol is described with enough detail to guarantee its reproducibility and the literature review is pertinent and exhaustive. The evidence presented is solid and reliable and it fully supports the conclusions reported. I have no major comments, just a very few minor suggestions to marginally improve clarity. Specifically: Line 121: "Two extraction methods were tested; one that..." could be replaced with: "Two extraction methods were tested: one that..." It could be useful to specify the pH of some of the buffers and solutions used for sample preparation, e.g. ABC. On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-181433.R2 entitled "A proteomic method to extract, concentrate, digest, and enrich peptides from fossils with colored (humic) substances for mass spectrometry analyses" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
• Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-181433.R2 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
Please note that we cannot publish your manuscript without these end statements included. We have included a screenshot example of the end statements for reference. If you feel that a given heading is not relevant to your paper, please nevertheless include the heading and explicitly state that it is not relevant to your work.
Because the schedule for publication is very tight, it is a condition of publication that you submit the revised version of your manuscript before 19-Jul-2019. Please note that the revision deadline will expire at 00.00am on this date. If you do not think you will be able to meet this date please let me know immediately.
To revise your manuscript, log into https://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions". Under "Actions," click on "Create a Revision." You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you will be able to respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". You can use this to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the referees.
When uploading your revised files please make sure that you have: 1) A text file of the manuscript (tex, txt, rtf, docx or doc), references, tables (including captions) and figure captions. Do not upload a PDF as your "Main Document". 2) A separate electronic file of each figure (EPS or print-quality PDF preferred (either format should be produced directly from original creation package), or original software format) 3) Included a 100 word media summary of your paper when requested at submission. Please ensure you have entered correct contact details (email, institution and telephone) in your user account 4) Included the raw data to support the claims made in your paper. You can either include your data as electronic supplementary material or upload to a repository and include the relevant doi within your manuscript 5) All supplementary materials accompanying an accepted article will be treated as in their final form. Note that the Royal Society will neither edit nor typeset supplementary material and it will be hosted as provided. Please ensure that the supplementary material includes the paper details where possible (authors, article title, journal name).
Supplementary files will be published alongside the paper on the journal website and posted on the online figshare repository (https://figshare.com). The heading and legend provided for each supplementary file during the submission process will be used to create the figshare page, so please ensure these are accurate and informative so that your files can be found in searches. Files on figshare will be made available approximately one week before the accompanying article so that the supplementary material can be attributed a unique DOI.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) In the manuscript entitled "A proteomic method to extract, concentrate, digest, and enrich peptides from fossils with colored (humic) substances for mass spectrometry analyses", Schroeter and co-authors describe an protocol to extract ancient protein residues from bone samples highly infiltrated with coloured (humic) substances. As the authors point out, the challenge the method aims at addressing is significant. The study clearly describes how the method presented clearly leads to higher recoveries in terms of proteins peptides and PSMs. In the current version the manuscript is clearly readable, the experimental protocol is described with enough detail to guarantee its reproducibility and the literature review is pertinent and exhaustive. The evidence presented is solid and reliable and it fully supports the conclusions reported. I have no major comments, just a very few minor suggestions to marginally improve clarity. Specifically: Line 121: "Two extraction methods were tested; one that..." could be replaced with: "Two extraction methods were tested: one that..." It could be useful to specify the pH of some of the buffers and solutions used for sample preparation, e.g. ABC. I am pleased to inform you that your manuscript entitled "A proteomic method to extract, concentrate, digest, and enrich peptides from fossils with colored (humic) substances for mass spectrometry analyses" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published. We thank both reviewers for their comments and suggestions. Below, we have detailed the edits we've made to the manuscript in response to their suggestions. Additionally, we have uploaded a "tracked changes" version of the current manuscript that shows all of these changes in the document (as well a few minor typos we have corrected).

Reviewer: 1
Comments to the Author(s) Schroeter et al. build in the current manuscript on their earlier works in an effort to mitigate a potentially major issue in palaeoproteomics -the presence of large amounts of humics in fossil bone interfering during protein extraction and MS analysis. This is a relevant question to resolve, as is indicated by the recent literature on this topic as well.
The major issue with the manuscript is that an internal control is lacking. That is, claims of relevance made in the paper of enhanced protein extraction through either Method 1 and Method 2 refer to extractions and data presented in a previous paper (Cleland et al., 2016, PRSB; Cleland et al., 2012, Plos One). However, recent research in protein preservation has shown remarkable variation of within-bone protein preservation, necessitating that extraction comparisons are performed on homogenized bone powders. Further issues with comparing the enhanced results derive from an absence in protein or peptide quantification, differences in MS set-up, and column chemistry. Therefore, the main conclusion of the paper could be seen as suggestive.
We thank reviewer 1 for their comments. We agree that a direct comparison is needed to quantify the relative contributions the many differences between the old study and the new study each had to the increase in protein identification, and to account for intra-bone variation. However, the main contribution of this paper is that this combined method allows protein extracts dark with humics to be concentrated and clarified without losing low-abundance proteins (e.g., NCPs). Thus, to address the reviewer's concerns we have softened the language to shift focus from relative "improvement" over an older method, to the fact that this combined workflow allows sensitive analyses of low-abundance proteins in fossils containing abundant humics (as supported by its retrieval of 12 non-collagenous proteins). Additionally, we have added a statement acknowledging the limits of the comparison that can be made between the two methods.
Modifications to the text include: Abstract-We have changed "This method allows better and more sensitive analyses of lowabundance proteins in fossils containing humics" to "This workflow allows analyses of lowabundance proteins in fossils containing humics." Results-We have added the following sentence to the paragraph comparing the 2015 study with the current study: "A direct comparison of the HCl-ABC method employed in Cleland 2015 1 and this combined method is needed to quantify the relative contributions to the observed increase in efficiency from the various differences in their workflows (e.g., extraction buffers, MS instrument, column packing) and to fully account for possible intra-bone variation in protein preservation in this specimen. [2][3] Regardless, this combined workflow obtained 15 additional proteins previously unreported for this specimen, supporting its overall improvement in recovery." Conclusion-We have changed "Because this method allows better and more sensitive MS analyses of low-abundance proteins in fossils with a high humic content" to "Because this workflow allows MS analyses of low-abundance proteins in fossils with abundant humic content" Minor comments: -Lines 54-65. This is one massive sentence. Please break up in smaller sentences.
We have separated this list into individual sentences. That section now reads as follows: "For example, these substances interfere with colormetric assays used for protein quantitation (e.g., Bradford and bicinchoninic acid (BCA) assays), 8 preventing accurate measurement of protein for subsequent analyses. They also bind silver-stain, generating false-positives in SDS-PAGE gels, or can pre-stain gel lanes and obscure chemical stains applied to separated proteins. 9-10 They can contaminate the isotopic content of ancient bone and interfere with stable isotope analyses. 2,11 In mass spectrometry experiments, humic substances can cause ion-suppression during electrospray ionization (ESI), 3, 12 leading to either depressed signal intensity or no signal (regardless of the presence of protein in the sample). Further, they can clog analytical columns or spray tips during liquid chromatography, 3 causing unstable spray and/or the loss of samples, columns, or spray tips. Even when these substances do not clog analytical columns, they can generate a build-up of particulate matter on the ion optics, which can suppress or prevent signal during analyses of all subsequent samples, necessitating expensive and time-consuming instrument cleaning." -Lines 85-98. The four elements of the used extraction methods are presented as novel. However, each of these has already appeared in the palaeoproteomic literature at least once. To avoid the suggestion that all four elements are introduced here for the first time, references to relevant literature are justified.
To clarify that this is a method combined from elements previously utilized in paleoproteomics and adapted for humic removal, we have added the following sentence to the introduction: "Although the individual components of this method have been variously applied in previous paleoproteomic studies (e.g., non-demineralizing buffer, 4-5 FASP 6-7 ) their combination and adaptation for humic removal has not yet been explored." Addtionally, because during the course of this review a new paper was published discussing the removal of humics from fossil bones for proteomics, we have added the following sentence to the paragraph of the introduction that talks about previous methods to remove humics from protein extracts: "Cleland et al. 5 employed a method utilizing both a non-demineralizing buffer and magnetic beads to separate humics from proteins in unconcentrated protein extracts." -Line 96: The re-occurring claim that this bone specimen has a particularly high humic content requires quantification.
A quantitative comparison of the humic content of this moa fossil versus typical fossil specimens is beyond the scope of this paper. Thus, we have modified sentences in the abstract, introduction, and conclusion to avoid suggesting the humic content of this fossil is exceptionally high rather than simply abundant.
Modifications to the text include: • Title-We have changed "fossils with high humic content" to "fossils with abundant humics" • Abstract-We have changed the phrase "a moa fossil with very high humic content" to "a moa fossil dark with humic content" • Abstract-We have changed the phrase "in fossils with high humic content" to "fossils with humic content" • Introduction-We have changed the phrase "a moa fossil with high humic content" to "a moa fossil with humic content" • Conclusion-We have changed the phrase "protein extracts from a moa fossil with a very high humic content" to "protein extracts from a moa fossil inferred from their dark color to have a high humic content" • Conclusion-We have changed "fossils with high humic content" to "fossils with abundant humic content." -Line 126 vs line 147: You mention "a small piece", but later refer to approx. 250 mg., which is a large piece of bone, especially of this age. These two statements are in conflict, especially as "small" frequently refers to samples under 20mg in the isotope, collagen, and bone proteome literature. We have removed "small" from line 126, and now simply refer to "a piece of dark cortical bone." -Line 257: I think you mean smaller than 3 kDa, not larger than 3 kDa. We did indeed mean "less than." We have corrected this typo.
Reviewer: 2 Comments to the Author(s) It is suggested that the methods section of the article should be revised to improve its clarity. We thank reviewer 2 for this recommendation. Because the reviewer did not provide specific instructions or recommendations, to improve clarity, we constructed a supplemental table (Supplemental Table 1) that breaks down each phase of the extraction and sample preparation procedures into a series of steps. The location of each action (e.g., in tube, in filter), the reagent used and the volume of it added, the incubation time and temperature, the centrifugation time and speed, and the number of times an action is performed/repeated, are listed in a concise format, to complement the more detailed description provided in the text.
To direct readers to this Humic substances are break-down products of decaying organic matter that co-extract with 28 proteins from fossils. These substances are difficult to separate from proteins in solution, and 29 interfere with analyses of fossil proteomes. We introduce a method combining multiple recent 30 advances in extraction protocols to both concentrate proteins from fossil specimens with high 31 humic content, and remove humics, producing clean samples easily analyzed by mass 32 spectrometry (MS). This method includes: 1) a non-demineralizing extraction buffer that Humic substances are break-down products of decaying organic matter 1 that, to the frustration of 55 many protein researchers, co-extract with proteins from fossils (e.g., 2 ) and soil (e.g., 3 ). These 56 complex, hydrophilic, dark-colored substances have a large molecular weight range (< 10 kDa to 57 > 100 kDa), and are notoriously difficult to separate from proteins in solution. 1,[4][5][6][7] This causes a 58 host of problems for a number of down-stream chemical analyses when conducting 59 paleoproteomics of fossil tissues. For example, these substances interfere with colormetric assays 60 used for protein quantitation (e.g., Bradford and bicinchoninic acid (BCA) assays), 8 preventing 61 accurate measurement of protein for subsequent analyses. They also bind silver-stain, generating 62 false-positives in SDS-PAGE gels, or can pre-stain gel lanes and obscure chemical stains applied 63 to separated proteins. 9-10 They can contaminate the isotopic content of ancient bone and interfere 64 with stable isotope analyses. 2,11 In mass spectrometry experiments, humic substances can cause 65 ion-suppression during electrospray ionization (ESI), 3,12 leading to either depressed signal 66 intensity or no signal (regardless of the presence of protein in the sample). Further, they can clog 67 analytical columns or spray tips during liquid chromatography, 3  157 Two extraction methods were tested; one that used a single incubation in a non-demineralization 158 reagent 14 for protein solubilization, and one that incorporated a brief pre-treatment of the bone 159 powder with NaOH, followed by an extraction identical to the first in all other respects. NaOH 160 has been used to remove "pigments" from bone prior to collagen extraction 24 , humic acids from 161 archaeological bone 2 , or to improve the efficiency of some bone protein extraction methods. 15 162 Therefore, we tested this extraction method both with and without an NaOH pre-treatment step 163 to assess its usefulness in both removing humics and extracting peptides. Figure 1 illustrates the 164 basic workflow of the developed extraction procedure (NaOH pretreatment step not shown). 165 Supplementary Table 1  solubilized in 50 µL of 50 mM ABC (well-mixed with the sample in the filter by pumping the 218 pipette as described above). Samples were allowed to digest overnight in a humidity chamber at 219 37˚C in a heating oven. After digestion, filters were centrifuged at 14,000 rcf for 30 min. Then, 220 40 µL of 50 mM ABC were added to the filter, mixed with the pipette to suspend any sediment at the bottom, and samples were centrifuged again at 14,000 rcf. This step was then repeated 222 twice, and the final filtered sample of ~150 µL was transferred to a new 1.5 ml Protein LoBind 223 centrifugation tube and received 3 µL of formic acid (FA) to quench any further digestion and 224 precipitate any remaining acid-insoluble humics out of solution while allowing peptides to 225 remain suspended. 3 Samples were then frozen at -80˚C to await stage-tipping. virtually indistinguishable from blank controls that were processed using (initially) empty tubes 292 as samples ( Figure S2). Initial humic-containing supernatants (Figure 2, Row 1) became 293 increasing dark and opaque during concentration, and the flow-through during filtering was 294 markedly lighter than the portion of the supernatant held back in the filter (Figure 3). 295 Additionally, although 3 kDa MWCO, 500 µL Amicon Ultra filter units can concentrate 500 µL 296 samples into a ~30 µL volume (and did so in the negative controls), the in-filter volume of 297 concentrated 400/200/4 bone samples was approximately ~150 µL ( Figure 3). These 298 observations indicate that the humic substances that co-extracted with the protein from the bone 299 largely remained in the filter instead of passing through, which is consistent with the reported 300 size of these substances (e.g., mostly >10 kDa 3 ) and the findings of Szpack et al. 2 that pre-301 digestion ultrafiltration is not useful for separation of humics from protein extract. These humic 302 substances remained in the filter even after digestion using the FASP protocol, allowing 303 tryptically digested peptides (now ≤ 3 kDa) to be separated from these substances when passed 304 through the filter during centrifugation. This separation was indicated by the drastic reduction in 305 color and opaqueness between the concentrated samples before ( Figure 3) and after (e.g., Figure  306 2E-F) digestion. Notably, although NaOH pretreatment has been reported as an effective method of removing 316 humic substances from fossil bone, 2 the single NaOH pretreatment incubation employed here 317 was not effective at removing a substantial amount of humics from the moa bone prior to protein 318 solubilization, as evidenced by the pale color of this fraction (Figure 2A), and the lack of 319 difference in color between pretreated and non-pretreated 400/200/4 fractions ( Figure 2B-C). 320 This difference may be because Szpack et al. 2 applied the NaOH treatment to demineralized 321 bone, whereas we applied it directly to ground bone powder. Nearly all the proteins identified were recovered from both methods, which shared 18 of the 20 333 proteins ( Table 1). The exceptions were periostin, which was only found in the "NaOH-334 400/200/4" fraction of Method 1, and prolargin, which was found in the "400/200/4 only" 335 extract, or Method 2. However, because these proteins were both identified by a relatively low 336 number of PSMs from their respective fractions (3-4 PSMs; In fact, when the number of unique (non-overlapping) peptides and percent sequence coverage 341 identified from each protein is considered, the results from Methods 1 and 2 are nearly identical, 342 with only slight variations that do not consistently favor either method (Figure 4). This suggests 343 that both methods recover roughly the same subset of the moa bone proteome. 344

345
When the two fractions of Method 1 are considered separately, the "NaOH-400/200/4" fraction 346 contains all the proteomic diversity obtained by this method; analysis of the "NaOH" 347 pretreatment extract did not identify any unique proteins compared to the subsequent fraction 348 (Table 1). Although the "NaOH" fraction possessed a greater number of collagen I PSMs than 349 the NaOH-400/200/4 fraction (Table 1) Previous LC-MS/MS analyses of this fossil produced sequences of collagen I, collagen II, and 361 collagen V. 22 The current method resulted in 15 additional proteins, including 12 NCPs, which 362 ranged from 1.3% to 18.4% sequence coverage for a given protein (STable 1, Figure 4B). This represents a substantial improvement over the previous study, which used a more common HCl 364 demineralization followed by ammonium bicarbonate solubilization, and utilized an entire gram 365 of fossil tissue as opposed to 250 mg. 22 A direct comparison of the HCl-ABC method employed 366 in Cleland 2015 22 and this combined method is needed to quantify the relative contributions to 367 the observed increase in efficiency from the various differences in their workflows (e.g., 368 extraction buffers, MS instrument, column packing) and to fully account for possible intra-bone 369 variation in protein preservation in this specimen. [29][30] Regardless, this combined workflow 370 obtained 15 additional proteins previously unreported for this specimen, supporting its overall 371 improvement in recovery. 372

373
The design of this protocol as described here could potentially be modified to suit the needs of 374 individual studies. For example, we used conical 3 kDa MWCO ultrafilters (Milipore), to ensure 375 that small bone proteins or protein fragments were not potentially being lost. However, 376 researchers that utilize 10 kDa MWCO filters for their studies (e.g., 19-20 ) might consider using 377 flat-bottomed filters, as there is some evidence that flat-bottomed ultrafilters may produce better 378 results by eliminating the dead space in the bottom of the filter. 31 Additionally, larger centrifugal 379 filters (e.g., 2 ml or 4 ml) may be used to reduce the concentration time, though the depth of the 380 larger filters may be more difficult for performing FASP. concentrate protein extracts from a moa fossil inferred from their dark color to have a high humic 388 content, then remove those humics to allow LC-MS/MS analysis without mechanical or ionic 389 interference from these substances. These analyses produced peptides from 20 bone proteins, 15 390 of which are newly identified in this specimen versus previous analyses using a more standard 391 approach. 22 Because this workflow allows MS analyses of low-abundance proteins in fossils with 392 abundant humic content, it has the potential to widen the range of extinct organisms amenable to 393 MS analyses, and increase regions of the proteome we can explore. 394

395
Although NaOH has been shown to aid in the removal of humic substances from a fossil bone 396 pellet that has been demineralized with HCl prior to NaOH treatment, 2 we found that a version of 397 this method that incorporates a NaOH pre-treatment step to the protocol (Method 1) does not 398 remove more humics, recover more proteins, or recover a substantially greater portion of the 399 sequence of any protein than the method utilizing only 400/200/4 (Method 2). This indicates that 400 NaOH pretreatment is not effective for humic removal in methods that utilize non-401 demineralizing 14 solubilization reagents to avoid loss of NCPs in the demineralization fraction. 15 402 We therefore recommend Method 2, as it produces nearly identical results with less cost in terms 403 of time, supplies, and instrument fees. 404

DATA ACCESSIBILITY 406
All RAW mass spectrometry data and Scaffold results files are available at Dryad 407 (http://dx.doi.org/10.5061/dryad.3tv1523). 408 All data files can be reviewed at: https://datadryad.org/review?doi=doi:10.5061/dryad.3tv1523. 409 We thank W. Zheng for logistical support and T. Cleland and two anonymous reviewers for 428 helpful comments on earlier versions of this manuscript.  filters. Despite combining five tubes with high humic content, passing samples through the filter 537 after digestion removed a large portion of the high-weight humics from the smaller peptides. 538 (Row 3) Supernatants after stage tipping. Upon elution from the stage tips, samples were nearly 539 colorless, suggesting that most inferring humic substances have been removed. 540 541 Figure 3. Concentrated 400/200/4 extract, before FASP digestion. Extracts dark with humic 542 substances became increasingly dark and opaque during concentration, indicating that humic 543 substances were too large to pass through the filter and co-concentrated with proteins. After 544 proteolytic digestion, these humics remained in the filter while digested peptides passed through. 545 We thank both reviewers for their comments and suggestions. Below, we have detailed the edits we've made to the manuscript.

Reviewer: 2
There are inconsistencies between different statements in the materials and methods section. For example, on page 6 it is stated that the bone sample was divided into 10 aliquots of which 5 were treated with NaOH and then all 10 samples were treated with the 400/200/4 solution. But on the following page (p7) it is stated that there were three sample sets, one of which had NaOH but no 400/200/4 treatment, which is inconsistent with the description given on the preceding page. Figure 1 is of little help in undestanding the experimental protocol. Overall, this article if revised could make a useful contribution to the palaeoproteomics literature but as submitted I think that it fails to meet the journal's requirement that published work should be "scientifically sound, in which the methodology is rigorous and the conclusions fully supported by the data." We apologize for the lack of clarity on how our sample sets were obtained. First, we will explain here where we think the confusion arose from: This paper tests two different methods for extraction.
• Method 1: incubates 5 aliquots of bone in NaOH, then collects and pools that NaOH. Then, the 400/200/4 solution is added to the same 5 aliquots of NaOH-treated bone, which is then collected and pooled into a separate sample. This generates 2 types of samples; a NaOH sample that was incubated with fresh, untreated bone (because it was the first step), and a 400/200/4 sample that is derived from the same, now NaOH-treated bone. Overall, Method 1 generates two sample types (NaOH treatment and NaOH-400/200/4 treatment) and Method 2 only one sample type (400/200/4 treatment). This is how we generated 3 sample types from 2 methods, and this is how we have a sample that has NaOH with no 400/200/4-it is generated as part of a two step method in which the first step (NaOH) and the second (400/200/4) are analyzed separately. Importantly, the NaOH-400/200/4 sample generated by this method does not combine the NaOH fraction and the 400/200/4 fraction of Method 1, it is only labeled this way to distinguish the 400/200/4 sample from bone that has had a NaOH pretreatment from the 400/200/4 sample from bone that did not have the NaOH pre-treatment (Method 2).
We agree this has the potential to be confusing, and is not elucidated by the workflow figure we provided previously. Thus, to illustrate the origin of these three samples more plainly, we have added the following diagram and figure caption to the supplemental material: