TY - JOUR
T1 - Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method
AU - Fu, Lijuan
AU - Amato, Nicholas J.
AU - Wang, Pengcheng
AU - McGowan, Sara J.
AU - Niedernhofer, Laura J.
AU - Wang, Yinsheng
N1 - Publisher Copyright:
© 2015 American Chemical Society.
PY - 2015/8/4
Y1 - 2015/8/4
N2 - The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m5C), 2′-O-methylcytidine (Cm), N6-methyladenosine (m6A), and 2′-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m5C, Cm and Am are tissue-specific. In addition, the 2′-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m5C and m6A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m5C, Cm, and Am are significantly lower (by 6.5-43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m6A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.
AB - The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m5C), 2′-O-methylcytidine (Cm), N6-methyladenosine (m6A), and 2′-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m5C, Cm and Am are tissue-specific. In addition, the 2′-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m5C and m6A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m5C, Cm, and Am are significantly lower (by 6.5-43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m6A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.
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U2 - 10.1021/acs.analchem.5b00951
DO - 10.1021/acs.analchem.5b00951
M3 - Article
C2 - 26158405
AN - SCOPUS:84938674856
SN - 0003-2700
VL - 87
SP - 7653
EP - 7659
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 15
ER -