Dynamic in vivo mapping of the methylproteome using a chemoenzymatic approach
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Abstract
Dynamic protein post-translation methylation is essential for cellular function, highlighted by the essential role of methylation in transcriptional regulation and its aberrant dysregulation in diseases including cancer. This underscores the importance of cataloging the cellular methylproteome. However, comprehensive analysis of the methylproteome remains elusive due to limitations in current enrichment and analysis pipelines. Here, we employ an L-Methionine analogue, ProSeMet, that is chemoenzymatically converted to the SAM analogue ProSeAM in cells and mice to tag proteins with a biorthogonal alkyne that can be functionalized for global detection, selective enrichment, and LC-MS/MS identification. We identify 486 proteins known to be methylated and 221 proteins with novel methylation sites encompassing diverse cellular functions. Systemic ProSeMet delivery in mice pseudomethylates proteins across organ systems with blood-brain barrier penetrance. Leveraging this pipeline to define the cellular methylproteome may have broad applications for understanding the methylproteome in the context of disease.
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Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Ruchika Bajaj and Gary McDowell. Review synthesized by Bianca Melo Trovò.
This study demonstrates the utility of an L-Methionine analog - ProSeMet - to tag and enrich proteins which have residues that are methylated in vivo, ex vivo and in vitro. Furthermore, the study demonstrates that this can be used in combination with mass spectrometry to identify these sites. Overall this is a useful, well-verified and well-described approach that will be helpful for future identification and investigation of methylation sites.
Major comments
It would be helpful if the manuscript could additionally discuss the reversibility of methylation generally, and the reversibility of the modification of protein residues by the alkyne group …
Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Ruchika Bajaj and Gary McDowell. Review synthesized by Bianca Melo Trovò.
This study demonstrates the utility of an L-Methionine analog - ProSeMet - to tag and enrich proteins which have residues that are methylated in vivo, ex vivo and in vitro. Furthermore, the study demonstrates that this can be used in combination with mass spectrometry to identify these sites. Overall this is a useful, well-verified and well-described approach that will be helpful for future identification and investigation of methylation sites.
Major comments
It would be helpful if the manuscript could additionally discuss the reversibility of methylation generally, and the reversibility of the modification of protein residues by the alkyne group specifically, in the discussion, and whether that has any implications for their results. It may be that the dynamics of methylation and demethylation vary between the two; or it may be that they are the same - either way, that may affect how they suggest others use this method and interpret its results.
Perhaps related to the question of reversibility, it would be helpful if the manuscript would comment on whether these are “true” methylation sites or not; i.e. whether they consider all these methylation sites to be functional. Trying to determine this would be an interesting direction for future work, but for this study a reflection on whether these novel functional methylation sites are simply capable of being methylated, or are likely to be methylation sites that are meaningful biologically, would be helpful.
Results, ProSeMet competes with L-Met to pseudo methylate protein in the cytoplasm and nucleus: the manuscript claims that ProSeMet is not incorporated into newly synthesized proteins but rather converted to ProSeAM and used by native methyltransferases. There does appear to be some reduction in the labeling with ProSeMet on cycloheximide treatment in Figure 2D - could this suggest that it is incorporated into newly synthesized proteins as well as being converted to ProSeAM? If not, could the manuscript explain why not? This experiment clearly shows that in contrast to AHA labeling, there is still use of ProSeMet as a substrate when translation is inhibited; however, it is not clear how this demonstrates that it is not incorporated at all into newly synthesized proteins. If methyl has been incorporated in previously present proteins, perhaps this can be clarified in the text.
Results, ProSeMet competes with L-Met to pseudomethylate protein in the cytoplasm and nucleus: the conclusion that “Cell fractionation of the cytosolic and nuclear compartments followed by SDS-PAGE fluorescent analysis revealed no fluorescent labeling of the L-Met control” is correct but may be overstated as there appears to be some background in the cytosolic fraction.
Minor comments
- Introduction: Recommend including a mention to ProSeMet's permeability.
- Introduction, Figure 1: the last step with CuAAC and N3 labeling in the description of the Chemoenzymatic approach for metabolic MTase labeling is not clear. Please, add the description in the legend.
- Results, Figure 2D: the image suggests an overloaded gel, consider using an alternative gel image.
- Supplementary Material, Fig. S1: the data with L-met is only shown with T47D stacks.
- Supplementary Material, Fig. S3: please add the control for the no treatment condition.
- Results, Fig. 2A ‘ incubating for 30 m in L-Met free media’: Please confirm that the length of incubation was 30 minutes.
- Results, Enrichment of pseudo methylated proteins used to determine breadth of methyl proteome: Please provide some description for the SMARB1-deficient G401 cell line. Why smarb1 deficient?
- Results, Figure 3: Please define BP, MF, HP, NES, and label the x and y axes in panel D.
- Results, ProSeMet-directed pseudo methylation is detectable in vivo: Please, clarify if the administration was oral.
Comments on reporting
- Results, ProSeMet competes with L-Met to pseudo methylate protein in the cytoplasm and nucleus: Please verify the quantity reported: 5µg on SDS-PAGE gel seems low.
- Results, ProSeMet-directed pseudo methylation is detectable in vivo: the manuscript reports that “mice starved prior to ProSeMet injection had increased ProSeMet labeling in the heart, whereas mice fed prior to ProSeMet administration had increased labeling in the brain and lungs”. The error bars are large, it would be helpful to show the individual real data points for the graphs in Figure 4.
- Results, Figure 4C: please report the mathematical expression used to calculate the relative fluorescence.
- Supplementary Material, Fig. S7: please provide more details on the antibody employed.
Suggestions for future studies
Future studies could investigate the biological functionality of the novel methylation sites - but this is a great proof of principle.
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