A Novel Analysis of the Peptide Terminome Characterizes Dynamics of Proteolytic Regulation in Vertebrate Skeletal Muscle Under Severe Stress
Round 1
Reviewer 1 Report
Kominami et al. present a study in which they characterized peptide terminome in non -model vertebrate organism utilizing a combination of transcriptomics and proteomics approach together with novel approach linear regression model. In addition, they compare the proteolytic dynamics of muscle proteins under air exposure as severe stress using Horse Mackerel.
The identification of the dynamics of proteolytic regulation under stress using non-model /un-sequenced animals is of interest both to the stress response community and the community working with new model organisms. The transcriptomics and proteomics method is appropriate with respect to choose of instrumentation.
The question, study execution, and results are straightforward. However, few technical issues are either not discussed or not addressed. The authors must carefully address all my concerns before further consideration.
My Concerns:
1. Analysis Method: It is not clear how different replicates (RNAseq/proteomics) are used to identify the Proteases. Are only those proteases are taken into consideration that is identified in all the replicates? Please elaborate in the method section about the reproducibility of the replicates and how the data are considered in the analysis.
2. Table 3 has the top-ranked genes based on Fold change, but is there some statistical assessment of how significant these scores are (p-values/adjusted p values)?
3. Supplementary Fig 1 is missing.
4. Fig3 Peptide terminome analysis heat map will be more interpretable if the names of the enzymes are provided instead of the EC numbers.
5. Please provide the mass-spec acquisition (data dependent and independent) conditions in details such as resolution (MS1, MS2 for DDA), collision energy etc.
6. As this is a study on the un-sequenced animals of its first time, it is a rich source of data having time course information. The data has not yet been made publicly available. It should be available in the standard repository. If it has already been submitted please mention.
Author Response
Response to Reviewer 1
The authors wish to express our deep appreciation to the reviewer for reviewing our manuscript and giving the useful comments and suggestions.
For the specific comments
1. Analysis Method: It is not clear how different replicates (RNAseq/proteomics) are used to identify the Proteases. Are only those proteases are taken into consideration that is identified in all the replicates? Please elaborate in the method section about the reproducibility of the replicates and how the data are considered in the analysis.
>> We wish to thank the reviewer for this comment. As we have described in Results (Dynamics of proteolytic regulation), total 156 proteases (classified to the class of EC 3.4) were identified in the transcriptome and classified into 72 classes based on the EC number. We took the 45 proteases into consideration whose cleavage site specificities were obtained from the MEROPS database. There were a few changes in gene expression of the 45 proteases, but they are thought to be constantly expressed in T.japonicus muscle tissue. Thus we calculated the matrix Xp respectively for each group (Deacap, AirEx1, 5, and 10) with N=45. In order to indicate this, we have inserted a sentence as follows: ‘The matrix Xp was respectively calculated for each group (Deacap, AirEx1, 5, and 10) by combining the SWATH data of 3 individual N=45.’ in Method (Multiple linear regression model).
2. Table 3 has the top-ranked genes based on Fold change, but is there some statistical assessment of how significant these scores are (p-values/adjusted p values)?
>> We believe the reviewer is mistaken on this admittedly ambiguous point. Table 3 has fold changes of cleaved proteins, it has not gene expression. We named the left side column as “Top-hit gene definition in BLASTX” because the cleaved proteins were identified through BLAST search. The column name easily leads misunderstanding, thus we have renamed the column to “Cleaved protein”. As we have described in Method (SWATH data processing), each peptide peak area was obtained by the sum of fragment ion XICs from MS/MS data and the areas for multiple peptides per protein were summed to obtain protein areas. The protein areas output by SWATH analysis are not equal to absolute sum of cleaved protein amounts. The value decreases as peptide fragments are further degraded into amino acid monomers. Thus we consider it is inappropriate to evaluate the peak area of cleaved protein statistically and only compared them based on fold changes.
3. Supplementary Fig 1 is missing.
>> We have submitted Supplementary Fig 1 (FigS1.tiff).
4. Fig3 Peptide terminome analysis heat map will be more interpretable if the names of the enzymes are provided instead of the EC numbers.
>> We agree with the reviewer’s comment. We have added the names of the enzymes identified in T.japomuscle tissue to the EC numbers (Fig. 3).
5. Please provide the mass-spec acquisition (data dependent and independent) conditions in details such as resolution (MS1, MS2 for DDA), collision energy etc.
>> In accordance with the reviewer's comment, we have inserted the details in Method as the followings,’ The following parameters were set for all shotgun analysis: ion spray voltage floating 2300 V, DP 80 V, CE 10 V, GS1 20 psi, GS2 0 psi, CUR 20 psi, and temperature 150 °C.’ and ‘The following parameters were set for all swath analysis: ion spray voltage floating 2300 V, DP 80 V, CE 10 V, GS1 20 psi, GS2 0 psi, CUR 20 psi, and temperature 150 °C.’.
6. As this is a study on the un-sequenced animals of its first time, it is a rich source of data having time course information. The data has not yet been made publicly available. It should be available in the standard repository. If it has already been submitted please mention.
>> We appreciate the reviewer's suggestion on this point. We already deposited the mass spectrometry data to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the jPOST partner repository with the dataset identifier (PXD 011911). The raw fastq files were deposited in the Sequence Read Archives (SRA) of the National Center for Biotechnology Information (NCBI) under accession number SRR8543815, SRR8543816, SRR8543817 and SRR8543818 of Bioproject PRJNA521293 and Biosample SAMN04485566. Then, we inserted them in the text.
Reviewer 2 Report
This study investigated the dynamics of proteolytic regulation under severe stress condition in a non-model animal T. japonicus. Transcriptomic and quantitative peptidomic analysis were performed to characterize the regulation through a novel analysis of the peptide terminome. This novel approach seems to be useful as demonstrated by that proteins related to glycolysis and muscle contraction systems were highly cleaved into peptides immediately under the severe stress.
The age and size of fish samples should be described in the methods. And it is not clear whether each group of fish samples was analyzed individually or not.
Line 326: Please discuss why no significant increase was observed in the AirEx10 group compared with the Decap group.
Author Response
Response to Reviewer 2
The authors wish to express our deep appreciation to the reviewer for reviewing our manuscript and giving the useful comments and suggestions.
For the specific comments
1. The age and size of fish samples should be described in the methods. And it is not clear whether each group of fish samples was analyzed individually or not.
>> We appreciate the reviewer for pointing that out. We have inserted size of fish samples in Materials and Methods (Fish samples). We used fish cultured for a short period after catch. It is unfortunately difficult to determinate the age of fish by analysis of their otolith, thus we can not estimate the age of fish samples. Actually the age-dependent proteolysis (sarcopenia) is not necessary to be considered because the fish of ~20 cm in the body length is estimated as about 1 year old as reported by Yoda et al. (Fisheries science, 80(1), 61-68, 2014).
2. Line 326: Please discuss why no significant increase was observed in the AirEx10 group compared with the Decap group.
>> We wish to thank the reviewer for this comment. We have inserted discussion as follows: ‘It is suggested that degradation rates of transcripts exceeded generation rates with nuclear destruction due to excessive contraction. The chromatin condensation in T. japonicus muscle after 10 min of air exposure has already been observed by transmission electron microscope (data was not shown).’
