CAP-MAP: cap analysis protocol with minimal analyte processing, a rapid and sensitive approach to analysing mRNA cap structures

Eukaryotic messenger RNA (mRNA) is modified by the addition of an inverted guanosine cap to the 5′ triphosphate. The cap guanosine and initial transcribed nucleotides are further methylated by a series of cap methyltransferases to generate the mature cap structures which protect RNA from degradation and recruit proteins involved in RNA processing and translation. Research demonstrating that the cap methyltransferases are regulated has generated interest in determining the methylation status of the mRNA cap structures present in cells. Here, we present CAP-MAP: cap analysis protocol with minimal analyte processing, a rapid and sensitive method for detecting cap structures present in mRNA isolated from tissues or cultured cells.

value and retention time. Considering that each channel shows one single peak for building the normalization curve accurately, why there are 2 peaks in the GpppGm channel? Is this a contamination or I am missing something? In Figure3., there are 2 peaks in m7GpppAm/m7Gpppm6A channel, and each peak should be annotated to its corresponding cap structure: which peak is m7GpppAm and which m7Gpppm6A? Also, MRM values should be labeled at each x-axis of the 11 cap structures. If author can add information on m7GpppCm and m7GpppUm (maybe also m7GpppC and m7GpppU), it could be useful for users to follow. In Figure 5A, in m7GpppAm ~5 fmol/ug RNA vs. m7Gpppm6Am ~12 fmol/ug RNA, CAPAM converts most of m7GpppAm into m7Gpppm6Am. However, in GpppAm 0.1 fmol/ug RNA vs. Gpppm6Am ~0.02 fol/ug RNA (the same trend in Figure 6B), it seems that CAPAM methylation depends on cap m7G methylation? Does RNMT knockdown trigger a dramatic decrease of m6Am level in cell mRNA? Maybe some data and explanation can be added in figure 7 and discussion section. Relationship between RNMT and m6Am will be critical to know.

Review form: Reviewer 2
Recommendation Accept with minor revision (please list in comments)

Do you have any ethical concerns with this paper? No
Comments to the Author Galloway and co-workers have reported on the development of an LC-MS assay amenable at the quantification of the different mRNA caps in the transcriptome. In this assay, mRNA is specifically enriched from total RNA via its poly(A)-tail, digested to nucleoside monophosphates and the trinucleotide cap using nuclease P1 and directly submitted to LC-MS analysis. Addition of an internal standard allowed for direct quantification of eleven distinct cap analogues. Since the methylation status of mRNA caps is thought to be dynamic, the method presented in this work can help understand these dynamics under different cellular conditions and in different tissues.
Comments to the authors: • The authors mention the recently published work of the Dedon group which is conceptually akin to this work. [1] However, the reader might find it difficult to understand the distinct differences between these methods. Could the authors therefore further elaborate on the conceptual differences between their method and the work of Wang et al.?
• The concentrations of the caps detected in this work are about one order of magnitude lower compared to the concentrations detected by Wang et al. (i.e.: m7Gpppm6Am: 20 fmol/µg RNA vs 425 fmol/µg RNA [1], both in mouse C57BL/6 liver cells). Could the authors comment on these differences? • We wonder why the authors speculate on the retention time of the nucleoside monophosphates when they should be readily detectable in their LC-MS setup? • The authors used a PGC column for chromatographic separation of the caps and compared its performance to a HILIC column. Could the authors elaborate on the advantages of the PGC column used in this work? Does it offer better separation than a classical C18 column in a UHPLC setup at the given pH? We are pleased to inform you that your manuscript RSOB-19-0306 entitled "CAP-MAP: Cap Analysis Protocol with Minimal Analyte Processing, a rapid and sensitive approach to analysing mRNA cap structures" has been accepted by the Editor for publication in Open Biology. The reviewer(s) have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, we invite you to respond to the reviewer(s)' comments and revise your manuscript.
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Data accessibility section To ensure archived data are available to readers, authors should include a 'data accessibility' section immediately after the acknowledgements section. This should list the database and accession number for all data from the article that has been made publicly available, for instance: Comments to the Author(s) The authors report an efficient method for quantifying different mRNA cap structures, via LC-MS. It allows monitoring variation of these cap structure levels in diverse cell circumstances, including viral infection, cell stress, gene knockdown, human disease etc. The method can be quite useful for the scientific community. I have a few questions that will need to be addressed.
In Figure 1., guanosine base structure within the chemical structure of 2nd transcribed nucleotide is wrong, it should be a C=O double bond without protonation at O6. Considering that each channel shows one single peak for building the normalization curve accurately, why there are 2 peaks in the GpppGm channel? Is this a contamination or I am missing something? In Figure3., there are 2 peaks in m7GpppAm/m7Gpppm6A channel, and each peak should be annotated to its corresponding cap structure: which peak is m7GpppAm and which m7Gpppm6A? Also, MRM values should be labeled at each x-axis of the 11 cap structures. If author can add information on m7GpppCm and m7GpppUm (maybe also m7GpppC and m7GpppU), it could be useful for users to follow. In Figure 5A, in m7GpppAm ~5 fmol/ug RNA vs. m7Gpppm6Am ~12 fmol/ug RNA, CAPAM converts most of m7GpppAm into m7Gpppm6Am. However, in GpppAm 0.1 fmol/ug RNA vs. Gpppm6Am ~0.02 fol/ug RNA (the same trend in Figure 6B), it seems that CAPAM methylation depends on cap m7G methylation? Does RNMT knockdown trigger a dramatic decrease of m6Am level in cell mRNA? Maybe some data and explanation can be added in figure 7 and discussion section. Relationship between RNMT and m6Am will be critical to know.

Referee: 2
Comments to the Author(s) Galloway and co-workers have reported on the development of an LC-MS assay amenable at the quantification of the different mRNA caps in the transcriptome. In this assay, mRNA is specifically enriched from total RNA via its poly(A)-tail, digested to nucleoside monophosphates and the trinucleotide cap using nuclease P1 and directly submitted to LC-MS analysis. Addition of an internal standard allowed for direct quantification of eleven distinct cap analogues. Since the methylation status of mRNA caps is thought to be dynamic, the method presented in this work can help understand these dynamics under different cellular conditions and in different tissues.
Comments to the authors: • The authors mention the recently published work of the Dedon group which is conceptually akin to this work. [1] However, the reader might find it difficult to understand the distinct differences between these methods. Could the authors therefore further elaborate on the conceptual differences between their method and the work of Wang et al.?
• The concentrations of the caps detected in this work are about one order of magnitude lower compared to the concentrations detected by Wang et al. (i.e.: m7Gpppm6Am: 20 fmol/µg RNA vs 425 fmol/µg RNA [1], both in mouse C57BL/6 liver cells). Could the authors comment on these differences? • We wonder why the authors speculate on the retention time of the nucleoside monophosphates when they should be readily detectable in their LC-MS setup? • The authors used a PGC column for chromatographic separation of the caps and compared its performance to a HILIC column. Could the authors elaborate on the advantages of the PGC column used in this work? Does it offer better separation than a classical C18 column in a UHPLC setup at the given pH? • A table showing all the respective cap concentrations in all investigated tissues would serve as a nice summary and overview to the reader. Especially, since in the CD8 T and HeLa cells the concentrations of a few select and not all 11 caps are shown in the figures. (Additional experiment: Cap quantification in human CCRF-SB cells. Can m7Gpppm6Am be found? Wang et al. found m7Gpppm6Am to be amongst the most abundant caps, whereas in the mouse liver cells (also investigated in this study) its concentration was about 10-fold decreased compared to the most abundant caps. [1]) Comments to the editor: Overall, we highly recommend publishing this work if the aforementioned points are addressed.