In-Depth Characterization of Apoptosis N-Terminome Reveals a Link Between Caspase-3 Cleavage and Posttranslational N-Terminal Acetylation

  1. Rawad Hanna
  2. Andrey Rozenberg
  3. Layla Saied
  4. Daniel Ben-Yosef
  5. Tali Lavy
  6. Oded Kleifeld

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  1. Version published to 10.1016/j.mcpro.2023.100584
  2. Version published to 10.1101/2022.09.19.508487v4 on bioRxiv
  3. Version published to 10.1101/2022.09.19.508487v3 on bioRxiv
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    Reply to the reviewers

    Dear Editor,

    Please find below our detailed responses (in black font) to the Reviewer's comments (in blue). In addition, and to the request of Reviewer #1, we added a PDF file called “Reply to the reviewers MS data” that shows MS/MS and quantification information of representative peptides which were selected based on their (different) caspase/control abundance ratios. We thank the reviewers for their time and valuable comments.

    NOTE: our original reply includes several tables and graphs that were not incorporated into our reply shown below

    Reviewer #1

    Page 4 - In contrast to the hindrance of N-terminal amine ionization by Nt-acetyl groups concluded by the authors, previous studies reported an improved MS-scoring if α-amino-acetylated (tryptic) peptides by the higher numbers of b and y fragment ions observed as compared to α-amino-free (tryptic) peptides (e.g. (Staes et al., 2008)). It is rather the lack of any N-/C-terminal charged residue in case of Lys-N type N-termini which makes LATE less suitable for studying N-terminal protein acetylation.

    We thank the reviewer for this comment. In the HYTANE and LATE workflows, only peptides with modified N-termini (by dimethylation or acetylation) are observed after negative selection, hence we argue that the important comparison here is between Nt-acetylated peptides and Nt-dimethylated peptides with (as in HYTANE) or without basic residue (as in LATE). While we are aware of the study by Staes et al 2008 (PMID: 18318009), we do not believe it contradicts our claim as it discusses the difference between Nt-acetylated peptides and peptides with free N-termini.

    As we indicated in the manuscript (page 5 in the last sentence of 1st paragraph), we observed less overall peptide identifications in LATE, which was expected due the lack of basic C-term residue. The reduction of identification was more pronounced for Nt-acetylated peptides. However, this still does not exclude LATE as a useful tool for identification of such peptides.

    Of note, the overall fragmentation coverage we obtained by LATE and HYTANE for Nt-acetylated and Nt-dimethylated peptides was similar. See the figure below.

    Hence, following Cho et al 2016 (PMID: 26889926), we suggest that the difference in ionization of Nt-dimethylated peptides vs Nt-acetylated peptides is the more dominant factor in peptide identifications.

    Figure 1:relative Ion coverage for modified peptides in LATE and HYTANE

    Page 4 - Besides indication the retained N-termini with high relative caspase-3/control abundance ratio's as putative caspase-3 proteolytic products, also indicate that unique peptides were retained, as many such singletons were reported in previous (caspase-focussed) degradomics studies making use of differential proteomics (e.g. (Van Damme et al., 2005)). Therefore the cut-off ratio of 2 rather seems unsubstantiated, unless the cellular proteomes of so-called control cells were affected by caspase activation. As such, showing some representative MS-spectra of neo-N-termini would be informative.

    We thank the reviewer for this comment. We agree that caspase-3 cleavage generates many singletons. This is indeed what we observed in the in vitro experiment as shown in Figure 2B by the long straight lines at Log2(caspase-3/control) >10. We also add here histograms of the obtained ratios that we hope will make this clearer. We chose a cut-off of 2 due to the basal activity of proteases (including caspase-3) as we did not add caspase-3 inhibitors to the cell lysate. In addition, peptides derived from the putative caspase-3 cleavages in the in vitro experiment were required to be identified only in the caspase-3-treated samples (i.e. to appear only with the heavy labeling). Minor changes to Figure 3 legend have been introduced accordingly. As can be seen in the table below, with a cut-off ratio of 2 (Log2=1) and selection of cleavage sites after D or E we identified >98% of the cleavage sites that were identified only in the caspase-3 treated samples (column text in blue). This rate did not change when the cut-off was set to 8 (Log2=3). Therefore, we have chosen to maintain our selection criteria and cut-off ratio as used before for both experiments.

    __Figure 2: Histograms of Log2(Caspase/control) ratios indicating the large number of singleton peptides (marked with arrows) __

    Table 1: In vitro experiment selection ratio

    Method

    Cutoff

    Time

    Sites

    Sites identified only caspase-3 treated samples

    % of caspase-treated only sites (singleton)

    Sites D/E with light

    Sites after D/E no light

    % of singleton

    LATE

    Log2=1

    18H

    936

    906

    96.8%

    798

    786

    98.5%

    LATE

    Log2=2

    18H

    884

    866

    98.0%

    767

    759

    99.0%

    LATE

    Log2=3

    18H

    819

    810

    98.9%

    722

    716

    99.2%

    HYTANE

    Log2=1

    18H

    1186

    1159

    97.7%

    1037

    1032

    99.5%

    HYTANE

    Log2=2

    18H

    1128

    1110

    98.4%

    998

    993

    99.5%

    HYTANE

    Log2=3

    18H

    1035

    1025

    99.0%

    924

    919

    99.5%

    LATE

    Log2=1

    6H

    755

    732

    97.0%

    656

    645

    98.3%

    LATE

    Log2=2

    6H

    711

    700

    98.5%

    630

    623

    98.9%

    LATE

    Log2=3

    6H

    671

    666

    99.3%

    601

    597

    99.3%

    HYTANE

    Log2=1

    6H

    1028

    988

    96.1%

    899

    890

    99.0%

    HYTANE

    Log2=2

    6H

    955

    931

    97.5%

    851

    844

    99.2%

    HYTANE

    Log2=3

    6H

    882

    871

    98.8%

    795

    791

    99.5%

    LATE

    Log2=1

    1H

    445

    423

    95.1%

    380

    372

    97.9%

    LATE

    Log2=2

    1H

    411

    402

    97.8%

    361

    355

    98.3%

    LATE

    Log2=3

    1H

    386

    380

    98.4%

    344

    338

    98.3%

    HYTANE

    Log2=1

    1H

    593

    559

    94.3%

    513

    506

    98.6%

    HYTANE

    Log2=2

    1H

    544

    532

    97.8%

    488

    482

    98.8%

    HYTANE

    Log2=3

    1H

    508

    498

    98.0%

    461

    455

    98.7%

    In the cell-based experiments of caspase-3, we induced apoptosis on both cell types (over-expressing caspase-3 and the control). Therefore, in this case, as the reviewer has also mentioned, a cut-off of 2 is appropriate because the control cells are also affected by caspase activation. Following the reviewer’s request we have added (in a separate PDF file) several representative MS/MS spectra of neo-N-term peptides and their corresponding quantification data.

    Page 4 - replace 'without labelling of lysine residues (epsilon-amines)' to 'without notable labelling of lysine residues (epsilon-amines)', as residual labelling of lysine side-chains was observed. Also in case of the latter, do note that reduced MS-ionization potential might impact labelling efficiency calculation, and chromatographic detection of labelling efficiency should be considered to conclusify this finding.

    We thank the reviewer for this comment. We have changed the sentence as requested (Page 4 marked in red). Regarding the labeling efficiency calculations, it is true that ionization potential might affect them. We used a common way to test this aspect (see e.g. Hurtado Silva *et al *2019 (PMID: 30934878)) and we are not aware of any reduction in ionization potential following lysine dimethylation. Although we did not study this aspect thoroughly, we frequently observe the opposite: that dimethylation improves MS detections.

    Page 6 - The experimental setup comparing caspase-3 overexpressing and ABT-199 induced versus ABT-199 induced cells will be highly biased as it will not be able to detect efficient caspase-3 cleavages (Plasman et al., 2011), as such cleavage events are complete and thus do not require any additional overexpressed capase-3. I see this as an important flaw and the authors should demonstrate that the list also includes efficient caspase-3 cleavages.

    We thank the reviewer for highlighting this important aspect. We agree that with our setup, we can miss some efficient cleavages of caspases-3. We acknowledged this caveat in the original text (page 6), but chose to perform our experiments this way in order to highlight cleavages that are affected by caspase-3 expression. To address the reviewer’s comment we have added new experiment and data on caspase cleavages that occur following ABT-199 treatment in HCT116 cells without overexpression of caspase-3. The focus of this experiment was on the relatively short time points following the ABT-199 treatment when no cell death is observed based on XTT assay (see Supplement Figure 6B). This experiment was used to prove that neo-Nt-acetylation of NACA is an early event in apoptosis (Figure 5 E-F page 12). We also used this experiment as an indication of the appearance of efficient cleavages. As can be seen from Supplement Table S10, if we consider all 3 time points of the ABT-199 treatment, we quantified 106 cleavages with free neo-Nt that were cleavages after D and were identified only in the ABT-treated samples. We refer to such cleavages, which appeared prior to noticeable cell death, as "efficient cleavages". Out of these efficient cleavages, 82 were also identified and quantified in the cell-based experiment with overexpression of caspase-3. Twenty efficient cleavages show a high ratio (≥2) in both experiments. Fifty six efficient cleavages had a high ratio in the new experiment and ratio below 2 in the cell-based experiment with overexpression of caspase-3. This supports our original claim regarding efficient cleavages and addresses the reviewer’s concern regarding our ability to identify efficient caspase-3 cleavages with the experimental setup of HCT116 cells overexpressing caspase-3.

    Page 12 - The setup doesn't permit ORF N-terminal stability per se, rather the cleavage susceptibly of N-termini holding (a) putative caspase-3 cleavage site(s). Please adjust accordingly. Again since the setup might have missed efficient cleavages, the assessment might be biased.

    Thanks for the comment. As requested, the word “stability” has been deleted. As discussed above, we demonstrate that our setup does allow the identification of efficient cleavages and hence our basis for believing that the assessment is not biased. Please also refer to our reply to the next comment.

    The claim that Nt-acetylation is protective for caspase-3 cleavage should be validated by monitoring cleavage efficiency of an Nt-acetylated versus an Nt-free variant (e.g. by introducing a Pro residue at AA position 2, or comparing cleavage efficiencies in corresponding NAT knockdown versus control cells) of an identified caspase substrate (i.e. a substrate holding a caspase-3 cleavage site in its N-terminal sequence) versus its Nt-free counterpart

    Thanks for raising this point. The reviewer's suggestions have some caveats: a mutation at a protein’s N-terminus in order to generate an Nt-free variant can alter its stability or function and NAT knockdown might have a profound biological impact on the cells. Therefore we chose a different way to study this aspect by selecting from our data ORF N-terminal peptides that were identified with both free N-termini and acetylated N-termini (i.e. the same peptide was identified in some PSMs as acetylated and in other as dimethylated). We managed to find 136 ORF N-terminal peptides that were quantified in both forms, and out of these, 122 contained Asp or Glu residues (the putative caspase cleavage motifs). We added the comparison of the abundance ratios of these peptides in Figure 4C (see also below). It shows a remarkable difference between the groups when the Nt-acetylated peptides ratios did not change as a result of caspase-3 overexpression while the peptides with free Nt were identified mostly in the control cells (negative Log2(caspase-3/control)). Comparison of the 14 ORF Nt-peptides that do not have Glu or Asp in their sequence shows no difference (see below).

    Figure 3: Abundance ratio distributions of the ORF Nt peptides identified with both Nt-acetylated and free Nt in HCT116 cells overexpressing caspase-3 and in the control. A. Comparison of peptides that contain putative caspase cleavage in their sequence (D or E) B. comparison of peptides without putative caspase cleavage

    These results provide additional support for the notion of the protective or shielding effect of Nt-acetylation against caspase-3 cleavage.

    Page 12 - Since post-translational Nt-acetylation of neo-N-termini could be reproduced in vitro in the non-dialyzed sample, enzymatic over chemical Nt-acetylation should be demonstrated (e.g. by the use of a (bisubstrate) NAT inhibitor).

    We think this is an interesting idea for future work. Yet, in our opinion, the fact that only very few neo-Nt-acetylated peptides were affected in vitro and that a similar trend of few selected neo-Nt-acetylation targets was shown in the cell-based experiments indicates that this process is enzymatic and not chemical in nature.

    Other concerns:

    Abstract - The abstracts holds complex/incorrect sentence constructions (e.g. simply indicate 'Protein N-termini', '... undergo ... processing by proteases' (currently: 'not be processed by proteases').

    Thanks for pointing this out. We have changed the abstract accordingly.

    Abstract - 'To expand the coverage of the N-terminome' only applies when this is used in conjunction with other negative enrichment strategies as by itself, LATE doesn't intrinsically provide a better coverage of the N-terminome (this is also noted at page 2).

    We thank the reviewer for pointing this out. We have changed the abstract accordingly.

    Change 'that cannot be identified by other methods' to 'that cannot be identified by other negative selection methods'

    Thanks for pointing this out. We believe that our description here is appropriate as we explicitly state “some of which cannot be identified by other methods”.

    Page 1 - Suggestion to change 'Proteases are typically described as degradative enzymes' to 'Proteases used to be described as degradative enzymes'

    Changed as suggested.

    Page 1 - Not really correct how written; 'N-terminomics methods highlight the N-terminal fragment of every protein (N-terminome)'

    Changed as suggested.

    Page 2 - Positive selection techniques .... Enrichment of unblocked (or Nt-free) N-termini

    We are not sure what the reviewer had in mind here but have added the text in the brackets

    Page 2 - Besides altering charge, Nt-acetylation also alters hydrophobicity ...

    Changed as suggested.

    Page 2 - remove 'to better chart'

    Changed as suggested.

    Page 2 etc. - Do note that caspase-3 can potentially activate downstream caspases in vitro

    Following this comment, we have added a sentence on Page 5 with this reservation

    Page 3 - functional crosstalk between proteolysis and neo-Nt-acetylation has already been demonstrated in the case of co-translational acting methionine aminopeptidases and chloroplast N-terminal acetyltransferases. Adjust accordingly.

    We thank the reviewer for highlighting this aspect, although we used the term “neo-Nt-acetylation” which we used to mark that this is not the common (co-translational) acetylation. To assure that this is more clear we have added the words “post-translational” to better define the novelty of our findings.

    Page 3 - when discussing the identification of ORF N-termini, note that some of the strategies of which note when used to enrich for in vivo blocked N-termini, can also be used without blocking/labelling of Lys residues, and thus trypsin will also result in Lys-ending peptides. This is important to consider in this context.

    Following the reviewer's remark we have changed the sentence so it now states: “Many of these N-terminomics methods……”

    Page 3 - remove the following sentence part; '... or run individually which can be useful for quantifying naturally modified N-termini.', since also a differential/labelled proteomics setup enables such assessment. Related to this, the authors should comment on the observation that much fewer (i.e. less than 40%) Nt-acetylated N-termini were identified by LATE as compared to HYTANE. How is this reflected in the number of PSMs? Probably these difference are further intensified when considering PSMs.

    We have changed the sentence as suggested.

    Regarding the reduction of Nt-acetylation, we thank the reviewer for this question as it led us to find typos in the numbers in Figure 1E which are now corrected. These typos did not change the overall observation that with LATE we identify fewer Nt-acetylated peptides than Nt-free (dimethylated) peptides. As the reviewer anticipated (see below), the reduction in LATE-based “contribution” to the identification of Nt-acetylated peptides as opposed to the identification of dimethylated peptides, is pronounced when considering PSMs but this is not much different than the peptide-based data. Therefore, we prefer to keep the current presentation of Figure 1E.

    __Figure 4: Comparison of HACAT cells N-terminal peptides identification with LATE and HYTANE when considering peptide sequences and PSMs. Peptides identified with both methods are in green and those that are unique to one method are in blue. Shared peptides were determined based on the sequence of the first 7 amino acids of the identified peptides. A. comparison for peptides with dimethylated N-terminal (free Nt) B. comparison for Nt-acetylated peptides. __

    Page 6 - Informative to indicate how many of the in silico predicted putative DEVD P4-P1 cleavages were actually present in the list of 2049 putative cleavages identified.

    In our dataset, we identified 17 cleavages after DEVD motif. 11 were identified only with HYTANE, 3 were identified by both methods, and 3 more were identified only with LATE. Of note, it seems that in large-scale proteomic studies of apoptosis, the number of caspase cleavages after DEVD motif is quite low. For example, in the CASBAH database (PMID: 17273173__) __there are 10 reports of such cleavage out of 391 reported sites, and in DegraBase (PMID: 23264352) that combined many different apoptotic experiments there are 64 reported DEVD sites out of a total of 6896 P1-Asp sites.

    Page 6 - Unclear if any of the of 2049 putative cleavages, included non-canonical P1 cleavages besides the P1 Asp and Glu cleavages identified.

    These are 2049 putative cleavage sites with P1 Asp or Glu. We have changed the text to make it clearer.

    Page 6 - Were the 'regular' cells mock transfected?

    No. The control cells used for the cell-based experiments were the non-transfected cells from the same culture of HCT166. We chose this option to guarantee that exactly the same cells that were grown in the same dish went through the same FACS sorting as a control.

    Page 6 -Important to note that an ORF can have multiple N-termini besides neo-N-termini (e.g. in the case of alternative translation initiation)

    Thanks for the great point. We have added an indication if the neo-N-termini site has been reported as an alternative translation initiation site to all of the results of the cell-based experiments (Supplementary Tables S4, S5, S6, S9). We also changed the Figures and text accordingly. Our analysis of reported/unreported neo-N-temini is based on the TopFind database which includes information about alternative translation initiation sites from TISdb. Of note, since our focus is on caspase cleavages and we further select putative cleavages based on D/E in P1 and fold change, out of 973 peptides that we reported as putative caspase cleavage (Table S6) only one is in the vicinity of an alternative initiation site.

    Page 6 - The authors should be more careful with generalization when comparing LATE and HYTANE (and other degradomics approaches) as in this study LATE was only applied for the identification of caspase-3 neo-N-termini, which by its extended substrate specificity might hold specific features enabling the preferred detection by one technique over the other. Also note that as compared to less recent studies, evidently the MS instrument used is a key factor in the increase in cleavages reported in the current study.

    It is conceivable that caspase cleavage may differ from other proteases and thus theoretically work better with LATE, but we fail to see why this would also be the case for other N-terminomics method (like TAILS, Subtiligase, CoFRADIC, ChaFRADIC etc). We showed that LATE provides additional ORF Nt peptides identifications and demonstrated its effectiveness in E. coli (Supplement Figure S2) also, which has a proteome with a different amino acid composition to the human proteome. Furthermore, using LATE in the cell-based experiment led to the identification of many neo-Nt-peptides that do not match caspase cleavage patterns (as indicated for both HYATNE and LATE in Figures 3E and 3F). We reviewed the text again, and believe that we have used a fair description of the results especially when we compared them to previous studies.

    Page 9 - The authors should provide some info/supporting statistics in the text regarding the new putative substrates showing GO-enrichments (compared to which control?) similar to previously reported caspase-3 substrates.

    The results of the GO enrichment analysis are presented in Fig. S8 and details about how the test was performed are provided in the Materials & Methods. In the revised version, we are including the numerical data that include results of the statistical tests per GO term as Table S12. The enrichment analysis was performed with respect to the whole human proteome.

    Page 11 - Indicate that the 11 neo-N-terminal peptides of which note are the neo-Nt-peptides matching (signal peptide) cleavages indicated in the Uniprot database. Were any corresponding di-methylated neo-N-termini of these cleavages identified? In case of the 'other' proteolytic cleavages of which note, refer to these as not-annotated in UniProt.

    We thank the reviewer for pointing this out. We have added an indication that this analysis is based on UniProt annotations. Yes, all of the reported 11 neo-Nt-Acet peptides shown in Figure 4 were also found as neo-Nt-DiMet peptides.

    Page 11 - post-translational Nt-acetylation is abundant in plant and the responsible NAT has been identified, please reference these studies as well.

    We thank the reviewer for pointing this out regarding page 11. A relevant reference has been added in Page 11. In the discussion, we already referenced Nt-acetylation in plants in the discussion as well (see page 14).

    Page 12 - Define 'undoubtedly dependent on caspase-3 cleavage'

    We thank the reviewer for pointing this out. The word ‘undoubtedly’ has been deleted.

    Page 14 - The NAA30 discussion is not really relevant for the discussion of the post-translational Nt-acetylation of mitochondrial neo-N-termini.

    We thank the reviewer for pointing this out. This sentence has been deleted.

    Viewing the harsh in vitro caspase-3 cleavage condition used, namely 1 µg caspase 3 over 20 µg protein, the P1 specificities of all identified neo-N-termini should clearly be shown.

    The P1 specificities of all neo-N-termini found in the in vitro experiment are listed in the supplementary tables S2 and S3. For the reviewer’s convenience, we are providing the table with the P1 specificities below:

    Since acetylation of serine and threonine residues are reported forms of post-translational modification, and many so-called past-translational Nt-acetylated neo-N-termini harbour such AA residues in their N-terminal sequence, b-ion coverage for these neo-N-termini should be provided/inspected.

    We are not sure that we understand this comment. O-Acetylation of amino acids refers to their side chain. Since we are using Di-methylation labeling in both HYTANE and LATE, if we have a peptide with O-acetylated Ser or Thr at its first position, it is possible to distinguish it from the same peptide with Nt-acetylation by MS1 as illustrated in the following table for a hypothetical peptide SAAANPELKR (mass is MH+1)

    Regardless we include in the manuscript MS/MS spectra of NACA Neo-Nt-acetylated peptide by HYTANE and LATE

    __Reviewer #2 __

    Major suggestions:

    • The LATE method relies on digestion with LysN. Can the authors comment on the digestion efficiency of the samples where the LATE workflow was applied?

    The LysN digestion details that we used were based on vendor (Promega) instructions combined with details from the Nature Protocol paper by Giansanti *et al 2016 *(PMID: 27123950__)__ that describes optimized digestion protocol for LysN. We tested LysN efficiency in terms of the identification of missed cleavage and found that it performed very well with a missed-cleavage rate of

    • The authors state that the number of peptides with acetylated N-termini was lower compared with HYTANE. Yet, the Nt-acetylation can occur co-translationally in approximately 85% of human proteins.

    Did the authors consider optimizing the method (e.g. by fractionating the sample) for better identification of such peptides?

    We thank the reviewer for this important comment. We are certain that it is possible to improve the output of LATE by fractionation and/or optimization by changes to the LC gradient as it is well established for most, if not all, bottom-up proteomics methods. In this work, we concentrated more on the proof of concept of the methodology and hence chose to work without fractionation. We performed one attempt to optimize the LC gradient but found that the results were not significantly different, and we thus used the same LC-MS methods that have been optimized for trypsin.

    Regarding the reduced identification of Nt-acetylated peptides, as we state in the manuscript following Cho et al 2016 (PMID: 26889926), we believe that this is mainly due to the reduced ionization efficiency of Nt-acetylated peptides compared to Nt-dimethylated peptides which is more pronounced when a C-terminal positive charge is missing (due to LysN digestion).

    Also, were the results of the study compared with searches done using other proteomic pipelines (e.g. FragPipe)?

    Unfortunately, when we started this project, MS-Fragger did not support LysN as the digesting enzyme. At the time TPP also provided better visualization and quantification support than FragPipe. Recently, we found that MSFragger is faster while providing similar identifications but we are not convinced of the quantification output via FragPipe. In addition, we performed comparisons of Comet to X!Tandem and while the searches took longer than with Comet, the total number of IDs did not improve significantly.

    Can the authors provide details on the settings used for searches done in COMET, especially for the samples treated with LysN?

    The settings are provided in Table S10 in the supplementary information (Page 14 of the PDF file).

    "Fractions containing relatively pure caspase-3 were pooled together and dialyzed against 20 mM HEPES 7.5, and 80 mM NaCl. Aliquots of the protein were stored at -80{degree sign}C"

    o What exactly is meant by 'relatively pure'?

    We apologize for the inaccurate description. The relevant text has been updated (Page 17) and now explains that this was based on Coommasse stain SDS-PAGE.

    Minor suggestions:

    • Please check the link for the Github as this reviewer could not open it.

    We thank the reviewer for pointing this out. We corrected the link. In any case, the relevant scripts can be found here: https://github.com/OKLAB2016

    • Please correct the spelling.

    The manuscript was proofread.

    Comments regarding figures:

    • Figure 2:

    o All figures comparing LATE and HYTANE utilize color green for LATE. Yet, in figure 2G, HYTANE is depicted in green-like color. Please consider staying consistent with the color scheme.

    We thank the reviewer for this comment. Done as suggested.

    Reviewer #2 (Significance (Required)):

    Significance:

    • The LATE method provides an excellent way to study proteases in vitro or in cell-based experiments. It enables deep investigation of N-terminome based on a simple and cost-effective workflow that utilizes digestion with LysN followed by chemical derivatization of α-amines. This approach allows for the identification of N-terminal peptides that may escape detection by other N-terminomics methods. With LATE, proteases' cleavage sites that might not so far be reporter due to technical limitations, can be studied and characterized. Hence, LATE is a useful addition to the N-terminomic toolbox.

    We thank the reviewer for the positive comments and general assessment of LATE.

    __Reviewer #3 __

    In this manuscript, Hanna et al. report LATE, an N terminomics method similar to N-TAILS and HYTANE, with modifications that enhance or change coverages of the N-terminal proteome in proteomics datasets. LATE relies on selective N-terminal modification of protease-treated, LysN digested samples, enabling internal peptides to be depleted based on the presence of the unblocked lysine epsilon amine. Using LATE in comparison with HYTANE, the authors identified a large number of both known and unknown caspase-3 cleavage sites, both in vitro and in vivo. Because LATE enables identification of both proteolytic neo-N termini and natively blocked N termini such as those that are acetylated, the authors were able to discover a number of post-translationally acetylated proteolytic neo-N termini. This finding points to potential functional cross talk between apoptotic proteolysis and Nt-acetylation. Overall, this is a very nice manuscript that adds a valuable new tool to the N-terminal proteomics toolbox. However, the manuscript could be improved by addressing the following questions and comments.

    We thank the reviewer for this assessment.

    One of the benchmark points used to describe the need for a new technology such as LATE is the idea that there are 134 putative caspase-3 substrates in the human proteome, of which only about half can be identified based on ArgC cleavages. However, the 134 substrates seem to include only those that have the exact canonical DEVD motif. Many more substrates than this are already known for caspase-3. For example, >900 caspase-3 substrates were identified by Araya et al. alone. It might make more sense to apply a position-specific scoring matric to the human proteome to predict a maximum number of possible caspase-2 cleavage sites and how many would be expected to be identified using other technologies. Otherwise, please provide a rationale for why these 134 putative caspase-3 sites are representative.

    The reviewer is correct. Indeed, most of the identified caspase-3 cleavage are not exact matches to the DEVD motif. We used the DEVD as an example to illustrate the added value of using lysine-based digestion together with ArgC. We obtained a similar trend with some variations when we tested the feasibility of the identification of the human ORF Nt-peptides, E. coli ORF Nt-peptides and more. We are quite confident that any prediction will show a relatively similar distribution. To demonstrate this, we show here the relative contribution of each method for the identification of any peptide that begins after Asp in the human proteome.

    While the distributions are not identical, they are very similar, and the specific additions from LATE (LysN) are between 20% to 22% out of the total and it can help to expand the coverage by 42% to 45%.

    It is definitely plausible and have been previously demonstrated that selective N-terminal demethylation can be achieved under the right reaction conditions, and I do not doubt that it has been achieved here. However, I do not understand how the authors were able to conclude that alpha-amines are blocked with 95% efficiency and lysines are blocked at

    This is a very good point. The reviewer is correct and indeed we don’t have a way to establish if the dimethylation is on the side chain amine of lysine or on its N-terminal amine. A partial support for our claim is from labeling experiments that we (and others) conducted with tryptic and LysC peptides that clearly demonstrate that under the specified labeling conditions, 95% of the N-terminal amines are labeled and not the lysine side chain amines. However, at the end of the day, this does not change the outcome of LATE.

    Related to the above comment, Table S10 seems to indicate that MS/MS data from LATE were searched with dimethylation as a fixed modification at the N terminus. Were LATE samples searched with different parameters to generate Figure 1C? Are the dimethylated Ks identified mostly from missed cleavages and therefore not at the N terminus?

    We thank the reviewer for pointing this out. The search parameters used for the generation of Figure 1C have been added to Table S10. The reviewer is correct, the few dimethylated Ks identified in the search used for Figure 1C are mostly from missed cleavages.

    For both the in vitro and in vivo experiments, how many of the new caspase-3 cleavage sites occurred in proteins that were not previously known to be caspase substrates?

    In the in vitro experiments, we identified cleavages of 372 proteins that were not reported as caspase-3 substrates based on the databases we used as references. A line specifying this number was also added to text on page 7. In the cell-based experiment, we identified putative caspase-3 cleavages of 67 proteins that were not reported so far as caspase-3 substrates. This information has been added to the main text on page 10. We have added columns indicating the known/unreported protein substrates to Tables S2, S3, S4, S5, and S6.

    For the experiment in cells, can the authors explain the rationale for comparing cells in which apoptosis is induced with ABT-199 to ABT-199-treated cells with caspase-3 overexpression? What is the advantage over comparing ABT-199 treated cells to untreated cells

    Great question. An N-terminomics study of “common” apoptosis would lead mainly to the identification of effector caspases (caspase-3 and -7) substrates. Our aim was to focus mostly on the caspase-3 cleavages that occur in the cell during apoptosis. In choosing this gain-of-function approach we were motivated by the idea that it couldprovide new insights that would otherwise go undetected when using knockout or other loss-of-function approaches. The advantage of this system over comparing ABT-199 treated to non-treated cells (which we have now added as well) is that it can enhance the identification of caspase-3 specific cleavages.

    Can the authors discuss the timescale of cell death in ABT-199 treated cells vs. ABT-199 treated caspase-3-overexpressing cells. Ideally, data showing cell viability over time (e.g. Cell Titer Glo or MTT assays) would be presented, but if the authors could at least describe whether apoptosis is accelerated in the caspase-3 overexpressing cells, it would be helpful.

    Great suggestion. Following the reviewer’s suggestion we have characterized the effect of caspase-3 overexpression of the cells by XTT assay, and indeed caspase-3 overexpressing cells do show accelerated cell-death in response to ABT199 compared to non-transfected cells. These results are now presented as Supplement Figure S6B and are mentioned in the results section.

    The authors say that in their experimental design, they expect to see no difference between ABT-199 only and ABT-199/caspase-3 overexpression for substrates that are cleaved efficiently by endogenous caspases. If the new caspase-3 substrates are not cleaved efficiently by endogenous caspase-3, this seems to call into question their physiological relevance. Can the authors explain more thoroughly how these new substrates fit into the apoptotic program?

    We thank the reviewer for raising this issue. We are aware that our original cell-based experimental design may have some limitations, yet we chose this gain-of-function setup in order to identify caspase-3 substrates in a cell-based system. We believe that this setup does allow identification of substrates that are efficiently cleaved by endogenous caspase-3, such as cleavage and acetylation of NACA at Ser34 (and neo-Nt-acetylation after caspase-3 cleavage in general). To study the physiological relevance of the neo-Nt-acetylation, we have added to the revised manuscript a time-course N-terminomics characterization of early apoptosis events conducted in HCT116 cells (without caspase-3 overexpression). The results of these experiments are now shown in Figure 5C and also in the Supplementary Table

    The authors convincingly show that cleaved NACA is a neo-substrate for Nt-acetylation, suggesting functional crosstalk between proteolysis and acetylation. However, it is not clear if this acetylation event has a functional consequence, so it seems inaccurate to say at the top of page 3 that "This is the first demonstration of functional crosstalk between neo-Nt-acetylation and proteolytic pathways."

    The author is correct. We changed the text accordingly.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, Hanna et al. report LATE, an N terminomics method similar to N-TAILS and HYTANE, with modifications that enhance or change coverages of the N-terminal proteome in proteomics datasets. LATE relies on selective N-terminal modification of protease-treated, LysN digested samples, enabling internal peptides to be depleted based on the presence of the unblocked lysine epsilon amine. Using LATE in comparison with HYTANE, the authors identified a large number of both known and unknown caspase-3 cleavage sites, both in vitro and in vivo. Because LATE enables identification of both proteolytic neo-N termini and natively blocked N termini such as those that are acetylated, the authors were able to discover a number of post-translationally acetylated proteolytic neo-N termini. This finding points to potential functional cross talk between apoptotic proteolysis and Nt-acetylation. Overall, this is a very nice manuscript that adds a valuable new tool to the N-terminal proteomics toolbox. However, the manuscript could be improved by addressing the following questions and comments.

    1. One of the benchmark points used to describe the need for a new technology such as LATE is the idea that there are 134 putative caspase-3 substrates in the human proteome, of which only about half can be identified based on ArgC cleavages. However, the 134 substrates seem to include only those that have the exact canonical DEVD motif. Many more substrates than this are already known for caspase-3. For example, >900 caspase-3 substrates were identified by Araya et al. alone. It might make more sense to apply a position-specific scoring matric to the human proteome to predict a maximum number of possible caspase-2 cleavage sites and how many would be expected to be identified using other technologies. Otherwise, please provide a rationale for why these 134 putative caspase-3 sites are representative.
    2. It is definitely plausible and have been previously demonstrated that selective N-terminal demethylation can be achieved under the right reaction conditions, and I do not doubt that it has been achieved here. However, I do not understand how the authors were able to conclude that alpha-amines are blocked with 95% efficiency and lysines are blocked at <5%. This claim seems to be based on PSMs for each type of modification. However, in a LysN digested sample, we would expect the vast majority of peptides to begin with K and the vast majority of Ks to be found at the N terminus of a peptide. In this situation, how is it possible to distinguish whether demethylation has occurred on the alpha-amine or the epsilon-amine? With N-terminal K, all of the MS2 fragments containing the N-terminal a-amine would also contain the lysine epsilon-amine. The m/z values for the y-ions, b-ions, and a-ions containing this residue would be the same. I may be misunderstanding, so it would be helpful if the authors could explain how they are able to distinguish these.
    3. Related to the above comment, Table S10 seems to indicate that MS/MS data from LATE were searched with dimethylation as a fixed modification at the N terminus. Were LATE samples searched with different parameters to generate Figure 1C? Are the dimethylated Ks identified mostly from missed cleavages and therefore not at the N terminus?
    4. For both the in vitro and in vivo experiments, how many of the new caspase-3 cleavage sites occurred in proteins that were not previously known to be caspase substrates?
    5. For the experiment in cells, can the authors explain the rationale for comparing cells in which apoptosis is induced with ABT-199 to ABT-199-treated cells with caspase-3 overexpression? What is the advantage over comparing ABT-199 treated cells to untreated cells
    6. Can the authors discuss the timescale of cell death in ABT-199 treated cells vs. ABT-199 treated caspase-3-overexpressing cells. Ideally, data showing cell viability over time (e.g. Cell Titer Glo or MTT assays) would be presented, but if the authors could at least describe whether apoptosis is accelerated in the caspase-3 overexpressing cells, it would be helpful.
    7. The authors say that in their experimental design, they expect to see no difference between ABT-199 only and ABT-199/caspase-3 overexpression for substrates that are cleaved efficiently by endogenous caspases. If the new caspase-3 substrates are not cleaved efficiently by endogenous caspase-3, this seems to call into question their physiological relevance. Can the authors explain more thoroughly how these new substrates fit into the apoptotic program?
    8. The authors convincingly show that cleaved NACA is a neo-substrate for Nt-acetylation, suggesting functional crosstalk between proteolysis and acetylation. However, it is not clear if this acetylation event has a functional consequence, so it seems inaccurate to say at the top of page 3 that "This is the first demonstration of functional crosstalk between neo-Nt-acetylation and proteolytic pathways."

    Significance

    See above.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors present a novel N-terminal enrichment method named LATE (LysN Amino Terminal Enrichment) that utilizes chemical derivatization of α-amines that enables characterization of the N-terminome. Using LATE as well as the already established HYTANE method, Hanna et al conducted a study of caspase-3 mediated proteolysis both in vitro and in cell-based apoptosis experiments, which led to the discovery of new potential caspase-3 cleavages. The results are well presented and nicely highlight that LATE is an efficient and inexpensive method that can be used to identify cleavage sites that cannot be found by other N-terminomics workflows.

    Major suggestions:

    • The LATE method relies on digestion with LysN. Can the authors comment on the digestion efficiency of the samples where the LATE workflow was applied?
    • The authors state that the number of peptides with acetylated N-termini was lower compared with HYTANE. Yet, the Nt-acetylation can occur co-translationally in approximately 85% of human proteins. Did the authors consider optimizing the method (e.g. by fractionating the sample) for better identification of such peptides? Also, were the results of the study compared with searches done using other proteomic pipelines (e.g. FragPipe)?
    • Can the authors provide details on the settings used for searches done in COMET, especially for the samples treated with LysN?
    • "Fractions containing relatively pure caspase-3 were pooled together and dialyzed against 20 mM HEPES 7.5, and 80 mM NaCl. Aliquots of the protein were stored at -80{degree sign}C"
      • What exactly is meant by 'relatively pure'?

    Minor suggestions:

    • Please check the link for the Github as this reviewer could not open it.
    • Please correct the spelling. Comments regarding figures:
    • Figure 2:
      • All figures comparing LATE and HYTANE utilize color green for LATE. Yet, in figure 2G, HYTANE is depicted in green-like color. Please consider staying consistent with the color scheme.

    Significance

    • The LATE method provides an excellent way to study proteases in vitro or in cell-based experiments. It enables deep investigation of N-terminome based on a simple and cost-effective workflow that utilizes digestion with LysN followed by chemical derivatization of α-amines. This approach allows for the identification of N-terminal peptides that may escape detection by other N-terminomics methods. With LATE, proteases' cleavage sites that might not so far be reporter due to technical limitations, can be studied and characterized. Hence, LATE is a useful addition to the N-terminomic toolbox.
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    Referee #1

    Evidence, reproducibility and clarity

    Manuscript Reference: RC-2022-01676

    TITLE: In-depth characterization of apoptosis N-terminome reveals a link between caspase-3 cleavage and post-translational N-terminal acetylation By Rawad Hanna, Andrey Rozenberg, Daniel Ben-Yosef, Tali Lavy, and Oded Kleifeld

    Summary of key results:

    The manuscript "In-depth characterization of apoptosis N-terminome reveals a link between caspase-3 cleavage and post-translational N-terminal acetylation" by Rawad and co-authors reports on a negative enrichment strategy, named LysN Amino Terminal Enrichment (LATE) to perform N-terminome analysis, a strategy which complements the cohort of existing negative enrichment strategies thereby jointly permitting a more comprehensive capture of the (neo-)N-terminome by additionally enabling the capture of (neo-)N-termini with (semi-)Lys-N specificity. The authors provide preliminary evidence that Nt-acetylation is protective for a proteins' N-terminus to be cleaved by caspase-3 besides the occurence of putative post-translational Nt-acetylation occurring on neo-N-termini generated upon caspase-3 cleavage.

    Concerns:

    Page 4 - In contrast to the hindrance of N-terminal amine ionization by Nt-acetyl groups concluded by the authors, previous studies reported an improved MS-scoring if α-amino-acetylated (tryptic) peptides by the higher numbers of b and y fragment ions observed as compared to α-amino-free (tryptic) peptides (e.g. (Staes et al., 2008)). It is rather the lack of any N-/C-terminal charged residue in case of Lys-N type N-termini which makes LATE less suitable for studying N-terminal protein acetylation.

    Page 4 - Besides indication the retained N-termini with high relative caspase-3/control abundance ratio's as putative caspase-3 proteolytic products, also indicate that unique peptides were retained, as many such singletons were reported in previous (caspase-focussed) degradomics studies making use of differential proteomics (e.g. (Van Damme et al., 2005)). Therefore the cut-off ratio of 2 rather seems unsubstantiated, unless the cellular proteomes of so-called control cells were affected by caspase activation. As such, showing some representative MS-spectra of neo-N-termini would be informative.

    Page 4 - replace 'without labelling of lysine residues (epsilon-amines)' to 'without notable labelling of lysine residues (epsilon-amines)', as residual labelling of lysine side-chains was observed. Also in case of the latter, do note that reduced MS-ionization potential might impact labelling efficiency calculation, and chromatographic detection of labelling efficiency should be considered to conclusify this finding.

    Page 6 - The experimental setup comparing caspase-3 overexpressing and ABT-199 induced versus ABT-199 induced cells will be highly biased as it will not be able to detect efficient caspase-3 cleavages (Plasman et al., 2011), as such cleavage events are complete and thus do not require any additional overexpressed capase-3. I see this as an important flaw and the authors should demonstrate that the list also includes efficient caspase-3 cleavages.

    Page 12 - The setup doesn't permit ORF N-terminal stability per se, rather the cleavage susceptibly of N-termini holding (a) putative caspase-3 cleavage site(s). Please adjust accordingly. Again since the setup might have missed efficient cleavages, the assessment might be biased.

    The claim that Nt-acetylation is protective for caspase-3 cleavage should be validated by monitoring cleavage efficiency of an Nt-acetylated versus an Nt-free variant (e.g. by introducing a Pro residue at AA position 2, or comparing cleavage efficiencies in corresponding NAT knockdown versus control cells) of an identified caspase substrate (i.e. a substrate holding a caspase-3 cleavage site in its N-terminal sequence) versus its Nt-free counterpart

    Page 12 - Since post-translational Nt-acetylation of neo-N-termini could be reproduced in vitro in the non-dialyzed sample, enzymatic over chemical Nt-acetylation should be demonstrated (e.g. by the use of a (bisubstrate) NAT inhibitor).

    Other concerns:

    Abstract - The abstracts holds complex/incorrect sentence constructions (e.g. simply indicate 'Protein N-termini', '... undergo ... processing by proteases' (currently: 'not be processed by proteases').

    Abstract - 'To expand the coverage of the N-terminome' only applies when this is used in conjunction with other negative enrichment strategies as by itself, LATE doesn't intrinsically provide a better coverage of the N-terminome (this is also noted at page 2).

    Change 'that cannot be identified by other methods' to 'that cannot be identified by other negative selection methods'

    Page 1 - Suggestion to change 'Proteases are typically described as degradative enzymes' to 'Proteases used to be described as degradative enzymes'

    Page 1 - Not really correct how written; 'N-terminomics methods highlight the N-terminal fragment of every protein (N-terminome)'

    Page 2 - Positive selection techniques .... Enrichment of unblocked (or Nt-free) N-termini

    Page 2 - Besides altering charge, Nt-acetylation also alters hydrophobicity ...

    Page 2 - remove 'to better chart'

    Page 2 etc. - Do note that caspase-3 can potentially activate downstream caspases in vitro

    Page 3 - functional crosstalk between proteolysis and neo-Nt-acetylation has already been demonstrated in the case of co-translational acting methionine aminopeptidases and chloroplast N-terminal acetyltransferases. Adjust accordingly.

    Page 3 - when discussing the identification of ORF N-termini, note that some of the strategies of which note when used to enrich for in vivo blocked N-termini, can also be used without blocking/labelling of Lys residues, and thus trypsin will also result in Lys-ending peptides. This is important to consider in this context.

    Page 3 - remove the following sentence part; '... or run individually which can be useful for quantifying naturally modified N-termini.', since also a differential/labelled proteomics setup enables such assessment. Related to this, the authors should comment on the observation that much fewer (i.e. less than 40%) Nt-acetylated N-termini were identified by LATE as compared to HYTANE. How is this reflected in the number of PSMs? Probably these difference are further intensified when considering PSMs.

    Page 6 - Informative to indicate how many of the in silico predicted putative DEVD P4-P1 cleavages were actually present in the list of 2049 putative cleavages identified.

    Page 6 - Unclear if any of the of 2049 putative cleavages, included non-canonical P1 cleavages besides the P1 Asp and Glu cleavages identified.

    Page 6 - Were the 'regular' cells mock transfected?

    Page 6 -Important to note that an ORF can have multiple N-termini besides neo-N-termini (e.g. in the case of alternative translation initiation)

    Page 6 - The authors should be more careful with generalization when comparing LATE and HYTANE (and other degradomics approaches) as in this study LATE was only applied for the identification of caspase-3 neo-N-termini, which by its extended substrate specificity might hold specific features enabling the preferred detection by one technique over the other. Also note that as compared to less recent studies, evidently the MS instrument used is a key factor in the increase in cleavages reported in the current study.

    Page 9 - The authors should provide some info/supporting statistics in the text regarding the new putative substrates showing GO-enrichments (compared to which control?) similar to previously reported caspase-3 substrates.

    Page 11 - Indicate that the 11 neo-N-terminal peptides of which note are the neo-Nt-peptides matching (signal peptide) cleavages indicated in the Uniprot database. Were any corresponding di-methylated neo-N-termini of these cleavages identified? In case of the 'other' proteolytic cleavages of which note, refer to these as not-annotated in UniProt.

    Page 11 - post-translational Nt-acetylation is abundant in plant and the responsible NAT has been identified, please reference these studies as well.

    Page 12 - Define 'undoubtedly dependent on caspase-3 cleavage'

    Page 14 - The NAA30 discussion is not really relevant for the discussion of the post-translational Nt-acetylation of mitochondrial neo-N-termini.

    Viewing the harsh in vitro caspase-3 cleavage condition used, namely 1 µg caspase 3 over 20 µg protein, the P1 specificities of all identified neo-N-termini should clearly be shown.

    Since acetylation of serine and threonine residues are reported forms of post-translational modification, and many so-called past-translational Nt-acetylated neo-N-termini harbour such AA residues in their N-terminal sequence, b-ion coverage for these neo-N-termini should be provided/inspected.

    References

    Plasman, K., Van Damme, P., Kaiserman, D., Impens, F., Demeyer, K., Helsens, K., . . . Gevaert, K. (2011). Probing the efficiency of proteolytic events by positional proteomics. Mol Cell Proteomics, 10(2), M110 003301. doi:M110.003301 [pii] 10.1074/mcp.M110.003301

    Staes, A., Van Damme, P., Helsens, K., Demol, H., Vandekerckhove, J., & Gevaert, K. (2008). Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC). Proteomics, 8(7), 1362-1370. doi:10.1002/pmic.200700950

    Van Damme, P., Martens, L., Van Damme, J., Hugelier, K., Staes, A., Vandekerckhove, J., & Gevaert, K. (2005). Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nat Methods, 2(10), 771-777. doi:nmeth792 [pii] 10.1038/nmeth792

    Significance

    The manuscript "In-depth characterization of apoptosis N-terminome reveals a link between caspase-3 cleavage and post-translational N-terminal acetylation" by Rawad and co-authors reports on a negative enrichment strategy, named LysN Amino Terminal Enrichment (LATE) to perform N-terminome analysis, a strategy which complements the cohort of existing negative enrichment strategies thereby jointly permitting a more comprehensive capture of the (neo-)N-terminome by additionally enabling the capture of (neo-)N-termini with (semi-)Lys-N specificity. The authors provide preliminary evidence that Nt-acetylation is protective for a proteins' N-terminus to be cleaved by caspase-3 besides the occurence of putative post-translational Nt-acetylation occurring on neo-N-termini generated upon caspase-3 cleavage.

  12. Version published to 10.1101/2022.09.19.508487v2 on bioRxiv
  13. Version published to 10.1101/2022.09.19.508487v1 on bioRxiv