TY - JOUR
T1 - High-throughput profiling of RNA modifications by ultra-performance liquid chromatography coupled to complementary mass spectrometry
T2 - Methods, quality control, and applications
AU - Huang, Gefei
AU - Zhang, Feng
AU - Xie, Dongying
AU - Ma, Yiming
AU - Wang, Pengxi
AU - Cao, Guodong
AU - Chen, Leijian
AU - Lin, Siyi
AU - Zhao, Zhongying
AU - Cai, Zongwei
N1 - Funding Information (Section snippets):
This work was supported by General Research Funds (N_HKBU201/18, HKBU12101520, HKBU12101522) from Hong Kong Research Grant Council, and Initiation Grant for Faculty Niche Research Areas RC-FNRA-IG/21–22/SCI/02 from Hong Kong Baptist University to ZZ.
Publisher Copyright:
© 2023 Elsevier B.V.
PY - 2023/10/1
Y1 - 2023/10/1
N2 - Although next-generation sequencing technology has been used to delineate RNA modifications in recent years, the paucity of appropriate converting reactions or specific antibodies impedes the accurate characterization and quantification of numerous RNA modifications, especially when these modifications demonstrate wide variations across developmental stages and cell types. In this study, we developed a high-throughput analytical platform coupling ultra-performance liquid chromatograph (UPLC) with complementary mass spectrometry (MS) to identify and quantify RNA modifications in both synthetic and biological samples. Sixty-four types of RNA modifications, including positional isomers and hypermodified ribonucleosides, were successfully monitored within a 16-min single run of UPLC–MS. Two independent methods to cross-validate the purity of RNA extracted from Caenorhabditis elegans (C. elegans) were developed using the coexisting C. elegans and Escherichia coli (E. coli) as a surveillance system. To test the validity of the method, we investigated the RNA modification landscape of three model organisms, C. elegans, E. coli, and Arabidopsis thaliana (A. thaliana). Both the identity and molarity of modified ribonucleosides markedly varied among the species. Moreover, our platform is not only useful for exploring the dynamics of RNA modifications in response to environmental cues (e.g., cold shock) but can also help with the identification of RNA-modifying enzymes in genetic studies. Cumulatively, our method presents a novel platform for the comprehensive analysis of RNA modifications, which will be of benefit to both analytical chemists involved in biomarker discovery and biologists conducting functional studies of RNA modifications.
AB - Although next-generation sequencing technology has been used to delineate RNA modifications in recent years, the paucity of appropriate converting reactions or specific antibodies impedes the accurate characterization and quantification of numerous RNA modifications, especially when these modifications demonstrate wide variations across developmental stages and cell types. In this study, we developed a high-throughput analytical platform coupling ultra-performance liquid chromatograph (UPLC) with complementary mass spectrometry (MS) to identify and quantify RNA modifications in both synthetic and biological samples. Sixty-four types of RNA modifications, including positional isomers and hypermodified ribonucleosides, were successfully monitored within a 16-min single run of UPLC–MS. Two independent methods to cross-validate the purity of RNA extracted from Caenorhabditis elegans (C. elegans) were developed using the coexisting C. elegans and Escherichia coli (E. coli) as a surveillance system. To test the validity of the method, we investigated the RNA modification landscape of three model organisms, C. elegans, E. coli, and Arabidopsis thaliana (A. thaliana). Both the identity and molarity of modified ribonucleosides markedly varied among the species. Moreover, our platform is not only useful for exploring the dynamics of RNA modifications in response to environmental cues (e.g., cold shock) but can also help with the identification of RNA-modifying enzymes in genetic studies. Cumulatively, our method presents a novel platform for the comprehensive analysis of RNA modifications, which will be of benefit to both analytical chemists involved in biomarker discovery and biologists conducting functional studies of RNA modifications.
KW - Caenorhabditis elegans
KW - Quality control
KW - Ribonucleoside
KW - RNA methylation
KW - RNA modifications
UR - http://www.scopus.com/inward/record.url?scp=85160433740&partnerID=8YFLogxK
U2 - 10.1016/j.talanta.2023.124697
DO - 10.1016/j.talanta.2023.124697
M3 - Journal article
AN - SCOPUS:85160433740
SN - 0039-9140
VL - 263
JO - Talanta
JF - Talanta
M1 - 124697
ER -