Peer review of: Fishing for newly synthesized proteins with phosphonate-handles (2020)


RR2
 REVIEWERS' COMMENTS:
Reviewer #1 (Remarks to the Author):
Reviewer #2 (Remarks to the Author):
  The authors addressed my questions and comments, and the manuscript is suitable for publication. I
 would have preferred to see an actual experimental comparison with desthiobiotin, but this is outside
 of the authors’ control due to current events. As a suggestion to the editor, other reviewer, and
 authors about the name issue, Pn is short for phosphonate, so PhosID could easily be switched to
 PnID.
  The authors have addressed most of the comments. However, there are still a few areas that need to
 be clarified.
 1. About major comment 3/4: It is difficult to carry out new experiments under current situation. But
 the authors should at least comment on the three phosphonate-containing probes about their potential
 advantages and disadvantages over each other. For example, it seems proteins are not fully
 denatured during the labeling process as described in the methods and indicated in Fig1C. Fig1B also
 shows that three P-handles are quite different in size. Are they subject to steric hindrance to different
 extent?
 The idea of adding a phosphonate handle to existing probes is not new. For example, CPT probes
 (phosphonate handle + succinimidyl iodoacetamide) were adopted to study free cysteine-containing
 peptides and protein redox regulation (https://doi.org/10.1016/j.cell.2020.02.012). One of the
 phosphonate handles in this reference is the same as what the authors used in this manuscript. If the
 authors intend to highlight the three phosphonate-containing probes instead of the existing concept of
 combining phosphonate handle and other probes, I think these three phosphonate-containing probes
 should be described in more details to facilitate the adoption by other people in the field.
 2. To address major comment 7, the authors added the explanation for “less than 2%” in
 supplementary figure 1 legend in the revised manuscript. And the newly added explanation says “less
 than 2.5%”. It’s a small issue but it’s better to be consistent (2% or 2.5%?), and it will be much more
 clear if the authors specify the 2.5% as what they did for other percentages in line 105 page 5, like
 “green:blue ratio in input”.
 3. About the major comment 8, the labeling time is 24 hr vs 3 weeks. Proteins are supposed to
 undergo turnovers over the longer period. Proteins are not just being synthesized as AHA-containing
 NSPs, NSPs are also degraded over time. The authors should elaborate more (and be specific) in the
 manuscript on what the high correlation means, and be specific about the number of proteins that are
 shared by the two labeling times and unique to either of the labeling time as well. In addition, it
 seems tab “PhosID - AHA (pulse) – mod” and tab “PhosID - AHA (stable) - mod” are identical in the
 corresponding table S4.
 4. The point of adding a Jurkat result similar to fig4C is to show the readers how the Jurkat data look
 like overall before comparing two cell lines. For example, do the two cell line data have the same
 depth? Do they have similar number of significant changes overall?
 5. The authors modified Fig1C and it is much better now. The illustration of a 3D protein structure is
 kept to represent proteins. The description about this part in the methods is a little fragmented. For
 example, line 492 page 15, cell pellets were resuspended in 1× PBS, pH 7.5, 1% sodiumdeoxycholate
 (SDC)), then diluted to 5 ug/ul in 1× PBS, pH 7.5. Then line 500 page 16, 500 ug total proteins
 containing 2 M Urea (final) in 1× PBS (pH 7.5) were used for chemistry reactions. Did the authors do a
 buffer exchange to switch from the lysis buffer to 2 M Urea? Some proteins remain undenatured in 2

 M Urea. Is this the reason why the 3D protein structure is kept? Is there a reason why not fully
 denatured proteins are used in the P-handle reactions? If it’s true that proteins are not fully denatured
 during P-handle reactions, it is better to make it clear in the manuscript.
 6. The authors added corrected p values to the IFN experiment. Please state in the methods how the
 FDR correction was performed.
 7. Fig S6, could the authors check on the “Log 10 scale” in the y-axis label? The y-axis is the number
 of unique peptides per protein, the numbers are quite big if they are on a log10 scale.
 8. Fig S7B, the title does not match the text in the figure legend.
 9. Fig4C, the authors did add the x-axis text (2-fold, 5-fold, etc). But they don’t match the x-axis
 labels (log2 ratio).