J Am Chem Soc. 2018 May 30: We enjoyed learning that in addition to the published by us and others work on the mutant methyonine tRNA synthase to bio-orthogonally and selectively tag de-novo sytnhesized proteins with Azido-Nor-Leucine (in vivo in het parabiosis, upon cell transplantation, etc.) one might also make mutations in tyrosine or phenylalanine tRNA synthases to tag the proteome with Azyde-conjugated tyrosine and phenylalanine. We find these findings very interesting, albeit the selectivity of the bio-orthogonal labeling might need improvement, as in Figure 2a, not only the GFP+ cells putatively expressing the mutant tyrosine or phenylalanine tRNA synthases, but also GFP negative cells, surprisingly, all show Click-chemistry fluorescence.
Most importantly, we did not find a strong evidence for the Mass Spectrometry results of Figures 3 and 4. First of all, DBCO affinity enriches but does not purify the Azide-tagged proteins and secondly, based on paper description, if in two MS runs at least one peptide containing AzydeY or AzydeF was detected, it was assumed that there was a protein tagged with Azyde-conjugated tyrosine and/or phenylalanine. In reality, such a low false discovery threshold produces false positives (in vitro and in vivo), e.g. from the cells that have been treated with AzideY or AzideF tags, but do not express the mutant forms of tRNA synthase(s). This control was sadly, not done. We suggest that the authors do the above mentioned negative controls to determine the tagged proteome; particularly, if they plan to follow on the studies by others and us to explore the age-specific, cancer-specific, etc. proteomes. When protein identification is optimized, one might also ask how different regiment of non-natural AA treatment (longer, for instance), M, Y and F content, protein length and abundance influence the effectiveness of the mMetRS L274G (previously and numerously published) as compared to the Y and F, reported here. Of note, supplementary Figure 6 does not show distinct positive AzidoAA treated versus noise AA treated populations (even on the selected Log scale), so it is unclear how or why some post DBCO signal was elected as positive or noise. As indicated above, the DBCO “noise” might resolve the false positive “AzidoY, F” hits on MS. Importantly, toxicity – cell viability studies were not done; the authors referenced previous literature, but did not address the fact that labeling was to saturation and with three different amino acid substitutions. In fact, such regiment is expected to cause toxicity and/or changes in properties of the proteins. This should be established.
Additionally, the statement:”Unlike prior studies, cell-type-specific bioorthogonal labeling via mutant aaRSs can distinguish tumor and host-secreted plasma proteins across a wide variety of
immunocompetent mouse models.” is inaccurate, as the same can be accomplished with mMetRS-ANL labeling. And the referencing of our paper as “Interest in adopting bioorthogonal labeling tools for cell and tissue-specific proteomics in mammals is growing” misrepresents the fact that the ref 33 reports on the method that this study paralleled.