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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this work, Kleinpenning et al. report a new strategy termed PhosID to purify proteins from complex 
mixtures using bioorthogonal noncanonical amino acid tagging (BONCAT). By using click chemistry to 
attach phosphonate handles onto metabolically incorporated azidohomoalanine residues, the authors 
show that they can effectively capture and release peptides containing the modified noncanonical 
amino acids. They synthesize the phosphonate tags with relative easy for both copper catalized and 
strain promoted azide alkyne cycloaddition, and they demonstrate that enrichments at the peptide 
level using PhosID have lower background signal that enriching at the protein level with biotin, which 
is a useful benefit. They demonstrate the utility of PhosID in an experiment that reveals temporal 
differences in proteome response between HeLa and Jurkat cells upon IFNγ stimulation. Overall, this is 
a well written manuscript and an important addition to the bioorthogonal chemistry toolkit. That said, 
there are enrichments at the peptide level using biotin (with anti-biotin antibodies) or desthiobiotin 
that, in theory, could accomplish the same goal. There is no doubt that PhosID is an improvement of 
protein-level enrichment with biotin followed by on-bead digestion, as their data shows here, but it 
would have been useful to see a straightforward comparison between these peptide-level enrichments 
and PhosID. I recognize that further experiments are difficult to conduct given current events 
worldwide, so I suggest that the authors at least discuss these other options in the manuscript at all 
points when comparing biotin enrichments to PhosID. I also have a few other comments to address, 
as indicated below. Even so, I believe this is a strong manuscript that can be ready for acceptance 
upon mostly minor revisions. 

• Discussion of desthiobiotin as a more transient/elutable version of the biotin/streptavidin interaction 
is necessary and warranted. I recognize that current events make it difficult to ask for more 
experiments, although a comparison of PhosID with desthiobiotin-based enrichment would be very 
interesting. That said, if the authors have any data on this on hand already, it would be a welcomed 
addition. One could imagine using desthiobiotin-NHS and desthiobiotin-alkyne to accomplish the same 
goal described here, even at the peptide enrichment level. Sentences such as “Conceptually, PhosID 
and biotin-streptavidin are distinct as PhosID enriches at the peptide level whereas the latter captures 
at the protein level”, page 5 would be nullified when consider desthiobiotin, which could enrich at the 
peptide level. Such sentences would also be nullified by the peptide-level biotin enrichment protocols 
cited in the introduction using anti-biotin antibodies. Quantitation comparisons would also be more 
straightforward using desthiobiotin or anti-biotin antibodies for enrichment at the peptide level. 
PhosID is a useful addition to the chemical biology toolbox regardless, but the caveat of desthiobiotin 
as an alternative should be addressed (benefits of not needing phosphatase treatment, etc). 

• Can the authors comment on the number of sialylated and phosphorylated glycopeptides that were 
enriched (a known phenomenon in IMAC enrichments)? This could be done for experiments where 
AHA was incorporated and when it was not (the background experiments in Figure 3B and 
Supplementary Figure 4). A simple count of spectra containing signature oxonium ions would be 
sufficient to comment on how many spectra in the enriched sample may be from species not from the 
PhosID specific peptides, given that phosphopeptides are largely eliminated (Supplementary Table 2). 
It would be valuable to know the number of interfering species that one could expect in a PhosID 
experiment, and if PNGaseF treatment should be added to the workflow along with phosphatase 
treatment. 

• Along the lines with the glycopeptide enrichment comment above, can the authors provide 
identifications of peptides/proteins that were seen in the background enrichment experiments where 
no AHA was included. I am mostly interested in identifications from the background PhosID 
enrichment – are peptides bound by IMAC here mostly glycosylated, or are there other proteins that 
could be filtered out of IMAC-enriched datasets? They provide the number of non-specific background 
proteins from the unlabeled HeLa lysate for the biotin enrichment (> 2000). Could those identifications 
be provided in as supplemental data along with the PhosID background IDs? Is digested streptavidin 
the major component of the total peptide mass from this background experiment, or are there other 
“sticky” background proteins that contribute substantially to the signal seen? A quick note about the 
abundance of streptavidin peptides in the background would be a valuable addition to the text. 

• It would also be interesting to have the protein identifications from Figure 3E as supplemental data 
to compare. 

• Instead of static cut-offs used for significant changes in the IFNγ (>2-fold, p < 0.05), I recommend 
the authors perform an multiple testing correction for significance, as is available in Perseus. It may 
not change the results much, but it is a more sound way of calculating significant changes. 

• Minor comment: if you are going to cite the BioSITe work, it is probably good to include the Carr et 
al. work on anti-biotin antibodies, as well: https://www.nature.com/articles/nmeth.4465 

• Minor comment, could the authors make the black borders for colors in figures less thick (or 
eliminate them entirely)? This would make it easier to see small percentages that are supposed to be 
a given color but currently look black because of the border lines. 



Reviewer #2 (Remarks to the Author): 

The total cellular proteome can nowadays be measured by MS with quite some success in depth and 
precision. However, there is still need for specific technologies focusing on selected smaller subsets of 
proteins. The manuscript by Kleinpenning et al. introduced three new phosphonate-handles. These 
probes selectively react with azide or alkyne containing biomolecules. Then the phosphonate-
containing biomolecules can be enriched with the widely used immobilized metal affinity 
chromatography (IMAC) in proteomics. The authors investigated newly synthesized proteins to 
benchmark these probes. Substitution of the Met in the cell culture medium using its analog AHA 
enables the incorporation of azides into newly synthesized proteins. The phosphonate-containing 
probes selectively react with the azides. Then IMAC was used to enrich the phosphonate-containing 
peptides for mass spectrometry analysis. The enrichment at peptide level is attractive in consideration 
of minimizing contaminants. The results showed these probe work. However, there are a few areas 
that should use elaboration/clarification to improve the manuscript, especially considering the scope 
and broad readership of Nature Communications. 

Major concerns: 

1. The title and abstract strongly indicate the probes are developed to investigate newly synthesized 
proteins. However, the introduction and discussion did not mention newly synthesized proteins, did 
not talk about previous tools (e.g. AHA)/methods for newly synthesized proteins. If the authors intend 
to present a new method/strategy for newly synthesized proteins, related research should be covered 
in the introduction and discussion. 

2. Theoretically the probes can be used to enrich any azide- or alkyne-containing biomolecules. If the 
investigation of newly synthesized proteins is just for benchmarking one of the applications of the 
probes, it’s better to clarify it in the manuscript and discuss other applications as well. 

3. As proof of concept, the authors showed that these probes work on functionalized BSA. But in HeLa 
experiment and INF experiment, it seems only one probe was demonstrated. It’s unclear how the 
other two probes perform in a real case scenario. 

4. What is the difference and similarity among these three probes in respective of performance, 
reactivity, specificity, stability, cost, etc? Are they biased towards a specific subset of peptides in cell 
line samples? How about the overlap between the identified peptides using different probes? It’s also 
confusing which probe is used in an experiment in the figures and text. Adding a couple words in the 
figures showing workflows should clarify it. 

5. In comparison with existing strategies, the authors adopted a biotin-based strategy. There is 
another widely used agarose-based strategy (alkyne agarose beads, as used in Ref14). The alkyne 
agarose strategy is supposed to perform better than the biotin-based strategy. What’s the advantages 
and disadvantage of PhosID in comparison of the alkyne agarose strategy? Have the authors done any 
experiments to compare them? 
6. A reference and a couple of sentences explaining “click-chemistry” in the introduction will be helpful 
considering the wide readership of Nature Communications 

7. The authors said “In addition, only a negligible amount (less than 2%) of the peptides were found 
to be labeled with a partially hydrolyzed probe synthesis byproduct (Supplementary Figure 2).” How 
was the 2% calculated? 

8. In Fig3D, the author said “In addition, PhosID does not bias for longer AHA labeling times, as pulse-
AHA and stable-AHA labeled samples were also found to be highly correlated (coefficient 0.99) (Figure 
3D).” It’s not clear what bias the authors are talking about here. The number of peptides or proteins? 
If the authors are talking about number, then the numbers in each group should also be specified. 
Should cells generate more newly synthesized protein in stable-AHA labeled samples (higher numbers 
of NSPs and higher abundance for a given NSP)? 

9. The authors studied the newly synthesized protein in both HeLa and Jurkat cell lines to benchmark 
one of the probes. 
a. Fig 4 only shows the Hela results. Results in Jurkat should also be described in the same way. 
b. A table summarizing the results in Fig 4c will be helpful. For example, how many peptides/proteins 
quantified, how many went up/down significantly. 

Minor points: 

1. The name “PhosID” is a little confusing. The first impression is this is a strategy for studying 
phosphorylation. 

2. Showing the AHA structure in Fig 3a can help with the understanding. 

3. Fig1C, the illustration of the PhosID is not clear enough. For example, under the leftmost arrow, it 
says “Digest”, but the arrow points to a full protein. In addition, a 3-D protein structure is confusing 
and misleading if the protein is already dissolved in lysis buffer. 

4. In the Methods, the last sentence in the “Hela spike-in experiments” paragraph, “supplementary 
Figure X” should be clarified. 

5. In the Methods, in the 4th row in the “Cell culture. AHA labeling and interferon stimulation” 
paragraph, clarify “PMID: 29669735”. 

6. In the methods, in the “Bioorthogonal chemistry reactions” paragraph, explain “MQ” 

7. In the methods, in the “PhosID sample preparation for LC-MS/MS” paragraph 
a. pH is missing for many buffers. For example, 50 mM ABC and 8 M Urea. Also specify in what 
solution the ABC and Urea Buffer are prepared. 
b. Describe the final conc. of DTT and IAM. 
c. Automated Fe(III)-IMAC workflow, provide essential description here instead of just citing two 
papers. 

8. In the methods, in the “LC-MS/MS” paragraph, some key parameters are missing or incomplete. 
For example, “dynamic exclusion duration of 16 sec” did not specify the times. Collision energy is also 
missing. 

9. Fig3A, fix the typo “Dephosphorlyate”. 

10. Fig3D, define the correlation, pearson or spearman? R or R-square? What is LC-MS intensity? Is it 
peptide or protein intensity? Or just use iBAQ intensity instead of LC-MS intensity since iBAQ intensity 
is explained in the methods. 

11. In the discussion it said the experiment in Fig4 used one (P-alkyne) of the probes. This should also 
be clearly indicated in Fig4a instead of using an ambiguous saying “PhosID protocol”. 

12. Fig S5, what’s the “log” in the y-axis label? 

13. Fig S7, define the correlation. R or R-squared? Pearson or spearman? 

14. Fig 4c, x axis label is missing. Texts are overlapping with each other. 

15. Table S4 
a. In the “content” tab, “T.test: t.test based on three technical replicates, two-tailed, paired”. Is it p 
value or t statistics? Or log-transformed p value? 
b. Fix the header of the last tab, they don’t match the content. 

16. Fig2A, define the black part (labeled by the by-product?) 

17. Fig5a and Fig5b, are ratios capped at 5 and -5?