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The analysis of DNA and RNA modifications is at the cutting edge of epigenetics-orientated biological sciences, and intensive interest is also emerging in the fields of environmental toxicology and health, pharmaceutics, and medicines. DNA 5-methylcytosine (5mC) is one well-known epigenetic modification that does not change the genetic coding information, but may regulate gene expression (1). Recently, it was found that 5mC can be oxidized as catalyzed by ten-eleven translocation protein family dioxygenases to form 5-hydroxymethylcytosine, which can be iteratively oxidized to form 5-formylcytosine and 5-carboxycytosine. These new modifications may be pertinent with active and passive DNA demethylation. Moreover, they may also play critical roles in mammalian cells by themselves. Several groups have also shown a new potential epigenetic mark, DNA N6-methyladenine modification, in high eukaryotes (2−4). Currently, 36 chemical modifications have been found in DNA (DNAmod database, last updated on August 28, 2016), and more than 120 modifications were found in RNA. Interestingly, the RNA modifications are also involved in many important biological processes; for example, N6-methyladenosine (m6A) in mRNA is involved in RNA stability, translation, splicing, transport and localization (5), and m5C in mRNA promotes mRNA export (6). Furthermore, modified nucleosides, as metabolites of DNA and RNA, are potential cancer biomarkers (7).
To understand the abundance, distribution, and functions of the diverse modifications of DNA and RNA, a number of methods have been developed, including bisulfite sequencing, antibody-based enrichment coupled next-generation sequencing, and liquid chromatography−mass spectrometry (LC−MS) (8). Among the known methods, ultrahigh-pressure liquid chromatography−tandem mass spectrometry (UHPLC−MS/MS) is generally one of the most popular methods for the identification and quantitation of DNA and RNA modifications at a global level. In this method, DNA and RNA are subjected to enzymatic digestion into normal and modified mononucleosides, and these mononucleosides are then separated by UHPLC followed by online-coupled MS/MS detection (9,10). Because of the highly efficient separation provided by UHPLC and structure-based MS quantification, UHPLC−MS/MS provides specific and sensitive detection of diverse modifications of DNA and RNA. It is also considered a “golden” method for accurate identification and quantification of diverse DNA and RNA modifications. To date, the development and applications of ultrasensitive and high-throughput UHPLC−MS/MS methods are of great interest to meet the requirement of the study of epigenetics. We summarize current concerns and future perspectives below.
Stationary Phases and Two-Dimensional Separations
Several types of stationary phases have been used to separate nucleosides. Reversed-phase high performance liquid chromatography (HPLC) is popularly used, but many polar nucleosides display poor retention on reversed-phase columns. Ion-pair reversed-phase HPLC is the technique of choice. Although this technique can improve the separation of polar nucleosides, most ion-pair reagents are not volatile and thus are incompatible to the online MS/MS detection. An alternative choice is hydrophilic-interaction chromatography (HILIC). In practice, irreversible adsorption of the polar nucleosides on the HILIC stationary phase was observed in our lab.
Some of the more than 150 (deoxy)nucleoside modifications are difficult to resolve because of the similarity of their structure and polarity. For example, 5mC and N4-methylcytosine (4mC), share the same ion pair for MS/MS quantitative analysis, but their retention on reversed-phase columns are similar. Two-dimensional (2D) chromatography is a promising separation technology for future profiling of these diverse (deoxy)nucleoside modifications.
Rapid and High-Throughput Analysis
We expected more than 10,000 samples per year in our lab. To deal with the large number of samples, we need make the analysis time as short as possible, while minimizing the matrix effects. Rapid and high-throughput UHPLC-MS/MS methods are highly desirable. In these methods, a stable isotope-labeled internal standard should be used to correct the impact of matrix effects.
Ultrasensitive MS/MS Detection
Some mononucleosides are hard to be ionized during electrospray ionization (ESI) and display poor MS/MS detection signal. We showed that ammonium bicarbonate can improve protonation of some nucleosides, and suppress the formation of MS signal-deteriorating metal complexes at the same time (11,12). Yuan and colleagues (13) showed a chemical derivatization of nucleosides for enhancing MS detection. The further improvement of MS detection is of great interest because it can significantly reduce the number of cells and DNA and RNA mass required for single-run analysis.
Online Digestion of DNA and RNA
In general, the digestion of DNA and RNA requires more than 2 h, making it tedious. In this process, some types of modified nucleosides are not stable, such as N6-hydroxymethyl adenine (hm6A) and N6-formyladenine (f6A), the intermediate products of m6A demethylation, with a half-life about 3 h in aqueous solution under physiological relevant conditions (14). If we want to analyze these nucleosides, we need to shorten both the sample preparation and analysis times. Our group constructed a three-enzyme cascade capillary monolithic bioreactor with deoxyribonuclease I, snake venom phosphodiesterase, and alkaline phosphatase on it, which can digest DNA into mono nucleosides when DNA samples flow through it (15). In future research, we will try to couple this capillary bioreactor with MS.
In summary, advanced UHPLC-MS/MS stimulates rapid developments on epigenetic research. On the other hand, the research in the field of epigenetics is also promoting the fast progress of UHPLC−MS/MS technologies to improve sensitivity, hyphenation with online digestion, and high-throughput analysis in the near future.
Hailin Wang and Weiyi Lai are with the State Key Laboratory of Environmental Chemistry and Ecotoxicology in the Research Center for Eco-Environmental Sciences at the Chinese Academy of Sciences in Beijing, China.