Abstract
Chemical modifications of transcripts with a 5′ cap occur in all organisms and function in many aspects of RNA metabolism. To facilitate analysis of RNA caps, we developed a systems-level mass spectrometry-based technique, CapQuant, for accurate and sensitive quantification of the cap epitranscriptome. The protocol includes the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by a 17 min chromatography-coupled tandem quadrupole mass spectrometry run for the identification and quantification of individual CNs. The total time required for the protocol can be up to 7 d. In this approach, 26 CNs can be quantified in eukaryotic poly(A)-tailed RNA, bacterial total RNA and viral RNA. This protocol can be modified to analyze other types of RNA and RNA from in vitro sources. CapQuant stands out from other methods in terms of superior specificity, sensitivity and accuracy, and it is not limited to individual caps nor does it require radiolabeling. Thanks to its unique capability of accurately and sensitively quantifying RNA caps on a systems level, CapQuant can reveal both the RNA cap landscape and the transcription start site distribution of capped RNA in a broad range of settings.
Key points
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CapQuant involves the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by chromatography-coupled tandem quadrupole mass spectrometry for the identification and quantification of individual CNs.
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CapQuant solves problems associated with other cap analysis tools, which are limited to individual cap structures, are poorly quantitative, lack sensitivity, require radioactive labeling and lack chemical specificity. It achieves high coverage with absolute quantification over a broad dynamic range starting at attomole levels.
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Data availability
Part of the data and supporting data were published in Nucleic Acids Research13. Supporting data and raw data can be found in Supplementary Information and deposited in the Chorus Project public repository (Project ID: 1809), respectively.
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Acknowledgements
We thank G. Walker and J. Chen from the Massachusetts Institute of Technology for generously sharing S. cerevisiae W1588-4C and human CCRF-SB cells, respectively. Financial support was provided by grants from the National Institutes of Health (ES022858 to P.C.D. and MH121072 and HG011563 to S.R.J.), the Singapore-MIT Alliance for Research and Technology with a grant from the National Research Foundation of Singapore, the National Natural Science Foundation of China (31960140 to J.W.), the Natural Science Foundation of Inner Mongolia Autonomous Region (2021JQ03 to J.W.), the Central Guiding Fund for Local Science and Technology Development (2020ZY0100 to J.W.), the Independent Project of State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock (SKL-IT-201830 to J.W.), the ‘Grassland Talents’ Program of Inner Mongolia Autonomous Region (to J.W.), the High-Level Talents Research Support Program of Inner Mongolia Autonomous Region (to J.W.), the ‘Steed Plan’ High-Level Talents Program of Inner Mongolia University (to J.W.), the Science and Technology Leading Talent Team Program of Inner Mongolia Autonomous Region (2022LJRC0009 to W. Hu from Inner Mongolia University and Fudan University), the Science and Technology Major Special Project of Inner Mongolia Autonomous Region (2020ZD0008 to Prof. Wei Hu from Inner Mongolia University and Fudan University) and the Nanyang Presidential Graduate Scholarship and the LKCMedicine Dean’s Postdoctoral Fellowship (to B.L.A.C.).
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Contributions
J.W. and P.C.D. conceived of CapQuant and designed the experiments. J.W., B.L.A.C. and P.C.D. developed the method, and wrote the first draft of the manuscript. J.W., B.L.A.C. and P.C.D. performed the cap analysis experiments and data analyses. Yong L. and T.K.L. performed yeast culturing. P.-Y.S. and H.D. were responsible for virus culturing, viral RNA isolation and synthesis of the GpppAm- and m7GpppAm-capped RNA oligos. S.R.J. provided the Gpppm6Am- and m7Gpppm6Am-capped RNA oligos and contributed insights and analysis. J.W. performed human and bacteria cell culturing, isolation of cell and tissue RNA, SEC purification of viral RNA, QC RNA quality experiments and data analyses, syntheses of all other (m7)GpppN(m)-capped RNA oligos and all 15N5-(m7)GpppN(m)-capped RNA oligos, purification of the unlabeled and labeled (m7)GpppN(m) CN standards, and determination of the IP of all labeled CN standards. D.L. provided the dengue NS5 methyltransferase that was used in the synthesis of the 15N5-Gpppm6Am-capped RNA oligo and contributed insights and analysis. X.-Y.F. and L.X. were responsible for the mouse experiments. B.L.A.C., P.C.D. and J. W. performed the cross-validation experiments and data analyses with insights from Z.L. Y.L. performed data analyses, prepared graphics, and edited the manuscript. All authors participated in the writing of the manuscript.
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J.W. is founder, executive director and general manager, and owns equity, in 中康鸿信. S.R.J. is cofounder of and advisor to, and owns equity in, Gotham Therapeutics. P.C.D. is cofounder and advisor to, and owns equity in, Hovana Inc., Proteomax Pte. Ltd., and Codomax Inc.
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Key reference using this protocol
Wang, J. et al. Nucleic Acids Res. 47, e130 (2019): https://doi.org/10.1093/nar/gkz751
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Supplementary Methods and Figs. 1–5.
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Wang, J., Chew, B.L.A., Lai, Y. et al. A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap epitranscriptome. Nat Protoc 18, 2671–2698 (2023). https://doi.org/10.1038/s41596-023-00857-0
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DOI: https://doi.org/10.1038/s41596-023-00857-0
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