TY - JOUR
T1 - Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications
AU - Xie, Yixuan
AU - Janssen, Kevin A.
AU - Scacchetti, Alessandro
AU - Porter, Elizabeth G.
AU - Lin, Zongtao
AU - Bonasio, Roberto
AU - Garcia, Benjamin A.
N1 - Funding Information:
The authors thank Juan J. Castillo and Siyu Chen (University of California, Davis) for their suggestions on the permethylation reaction. This work was supported by Grants (Nos. AI118891, CA196539, and AG031862) to B.A.G. from the NIH.
Publisher Copyright:
© 2022 American Chemical Society.
PY - 2022
Y1 - 2022
N2 - Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography−tandem mass spectrometry (LC-MS/MS)based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.
AB - Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography−tandem mass spectrometry (LC-MS/MS)based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.
UR - http://www.scopus.com/inward/record.url?scp=85131047103&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.2c00471
DO - 10.1021/acs.analchem.2c00471
M3 - Article
C2 - 35549217
AN - SCOPUS:85131047103
SN - 0003-2700
VL - 94
SP - 7246
EP - 7254
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 20
ER -