Lysine and Arginine Protein Post-translational Modifications by Enhanced DIA Libraries: Quantification in Murine Liver Disease
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###Reviewer #2:
This manuscript concerns the application of a narrowed mass window DIA method for simultaneous detection of modified (methylated, succinylated, acetylated Arg and Lys) and unmodified peptides in the same MS run. The authors use a combination of synthetic peptide libraries and immunoaffinity enriched samples to compare the performance of several mass windows, ultimately showing improved separation of modified and unmodified (precursor) peptidoforms using a 4 Da separation window. They apply this method with a modified site localization algorithm to identify modification sites that are differentially affected by hypo- and hypermethylation potential in mouse NASH models. These studies reveal potential connections between SAM levels and methylation potential with mRNA translation and acetylation levels. Overall, this work …
###Reviewer #2:
This manuscript concerns the application of a narrowed mass window DIA method for simultaneous detection of modified (methylated, succinylated, acetylated Arg and Lys) and unmodified peptides in the same MS run. The authors use a combination of synthetic peptide libraries and immunoaffinity enriched samples to compare the performance of several mass windows, ultimately showing improved separation of modified and unmodified (precursor) peptidoforms using a 4 Da separation window. They apply this method with a modified site localization algorithm to identify modification sites that are differentially affected by hypo- and hypermethylation potential in mouse NASH models. These studies reveal potential connections between SAM levels and methylation potential with mRNA translation and acetylation levels. Overall, this work presents a new methodology for simultaneous detection and quantitation of modified proteoforms without requiring parallel runs for enriched and unmodified protein detection. This methodology should be of interest to the proteomics community. Several of the mechanistic connections made in the NASH model are preliminary. There are several other aspects of the method presentation that should be addressed in the comments below.
Major Concerns/Comments:
The mechanistic jump from moderate alteration of methylation in three ribosomal proteins to causing decreased mRNA levels is not supported. The authors would need to add significantly more detail on where these modifications are and what quantitative changes are observed, as well as how these changes can affect the function of the protein of interest. Additionally, the claim that using the 4 Da DIA acquisition aids in understanding this mechanism should be expanded.
Similarly, the connections listed in the acetylation section are very tenuous. Specific proteins and deacetylases are listed and connected, but other relevant proteins that play redundant or counteracting roles are not considered. A more holistic presentation of sirtuins and hdacs should be included as they will collectively control the acetylation status. Finally, what is the conclusion of this section? That acetylation is lowered due to a series of effects leading to sirt3 mediated deacetylation? This should be supported experimentally if these claims are to be made.
Overall, the causal, rather than corrective relationships discussed on the sections focused on quantifying differential methylation/modification present in hypo/hypermethylated mouse models should be changed. For example, the authors make statements like "to determine the role that differential methylation potential plays in NASH...". The altered prevalence of sites is correlated with altered methylation potential, but these data do confirm they are playing a role in NASH. Statements like these should be adjusted.
Do the authors integrate information about cleaved peptides? This co-isolation issue is primarily an issue when exactly the same peptide +/- modification is close in chromatographic space. Yet the unmodified version of many of these target peptides will be cleaved by trypsin, creating a completely different peptide. How is this accounted for in data analysis?
The authors include a section on modifying the localization algorithm Thesaurus for the modifications studied here. Can the authors discuss these changes so the readers can assess whether these changes are appropriate and how they affect the altered performance?
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###Reviewer #1:
The manuscript by Robinson et al describes improvements to the DIA technique that are focused on enabling the quantitation of peptides bearing subtly different PTMs on lysine and arginine residues. The technique utilizes small DIA isolation windows to avoid co-isolation of precursor peptides whose m/z's are close (i.e. unmethylated vrs monomethylated or mono- vrs di-methylated, etc). The authors demonstrate that it can be utilized on unenriched samples which permits simultaneous assessment of changes to whole protein levels. Furthermore, they extend their localization algorithm (Thesaurus) to utilize these data and show POC by characterizing changes to PTMs in two mouse models of NASH.
The study represents quite a lot of work and it shows a high level of methodological sophistication, however it is quite narrow in scope. …
###Reviewer #1:
The manuscript by Robinson et al describes improvements to the DIA technique that are focused on enabling the quantitation of peptides bearing subtly different PTMs on lysine and arginine residues. The technique utilizes small DIA isolation windows to avoid co-isolation of precursor peptides whose m/z's are close (i.e. unmethylated vrs monomethylated or mono- vrs di-methylated, etc). The authors demonstrate that it can be utilized on unenriched samples which permits simultaneous assessment of changes to whole protein levels. Furthermore, they extend their localization algorithm (Thesaurus) to utilize these data and show POC by characterizing changes to PTMs in two mouse models of NASH.
The study represents quite a lot of work and it shows a high level of methodological sophistication, however it is quite narrow in scope. It will be of interest to mass-spectrometrists that utilize DIA, but not to a general audience.
Specific concerns:
The paper barely acknowledges the fact that peptides modified on lysine and arginine typically don't cleave efficiently with trypsin thereby resulting in missed cleavages. Thus most of the time it's quite simple to distinguish modified from unmodified without the need for narrow isolation windows.
DIA can be quite useful, but this reviewer cannot help but think that PRM might be more well-suited to detailed studies of peptidoforms with subtly different PTMs. If PRM is utilized, isolation windows can be as narrow as 1Da so the techniques employed in this manuscript are unnecessary.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 3 of the manuscript.
###Summary:
While the Reviewers were in agreement that your paper reports a useful method, they also felt that it was narrow in biological focus and of primary interest to those within the mass spectrometry-based proteomics community. The Reviewers also question whether the method offers substantial advantages over alternative approaches for analyzing Lys/Arg PTMs by MS-based proteomics.
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