Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

Simultaneous Detection of Adenosine-to-Inosine Editing and N6-Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling.

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) modifications are pivotal RNA modifications with widespread functional significance in physiological and pathological processes. Although significant effort has been dedicated to developing methodologies for identifying and quantifying these modifications, traditional approaches have often focused on each modification independently, neglecting the potential co-occurrence of A-to-I editing and m6A modifications at the same adenosine residues. This limitation has constrained our understanding of the intricate regulatory mechanisms governing RNA function and the interplay between different types of RNA modifications. To address this gap, we introduced an innovative technique called deamination-assisted reverse transcription stalling (DARTS), specifically designed for the simultaneous quantification of A-to-I editing and m6A at the same RNA sites. DARTS leverages the selective deamination activity of the engineered TadA-TadA8e protein, which converts adenosine residues to inosine, in combination with the unique property of Bst 2.0 DNA polymerase, which stalls when encountering inosine during reverse transcription. This approach enables the accurate quantification of A-to-I editing, m6A, and unmodified adenosine at identical RNA sites. The DARTS method is remarkable for its ability to directly quantify two distinct types of RNA modifications simultaneously, a capability that has remained largely unexplored in the field of RNA biology. By facilitating a comprehensive analysis of the co-occurrence and interaction between A-to-I editing and m6A modifications, DARTS opens new avenues for exploring the complex regulatory networks modulated by different RNA modifications.

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.

Site-specific quantification of Adenosine-to-Inosine RNA editing by Endonuclease-Mediated qPCR.

RNA molecules contain diverse modified nucleobases that play pivotal roles in numerous biological processes. Adenosine-to-inosine (A-to-I) RNA editing, one of the most prevalent RNA modifications in mammalian cells, is linked to a multitude of human diseases. To unveil the functions of A-to-I RNA editing, accurate quantification of inosine at specific sites is essential. In this study, we developed an endonuclease-mediated cleavage and real-time fluorescence quantitative PCR method for A-to-I RNA editing (EM-qPCR) to quantitatively analyze A-to-I RNA editing at a single site. By employing this method, we successfully quantified the levels of A-to-I RNA editing on various transfer RNA (tRNA) molecules at position 34 (I34) in mammalian cells with precision. Subsequently, this method was applied to tissues from sleep-deprived mice, revealing a notable alteration in the levels of I34 between sleep-deprived and control mice. The proposed method sets a precedent for the quantitative analysis of A-to-I RNA editing at specific sites, facilitating a deeper understanding of the biological implications of A-to-I RNA editing.

Formation and removal of 1,N6-dimethyladenosine in mammalian transfer RNA.

RNA molecules harbor diverse modifications that play important regulatory roles in a variety of biological processes. Over 150 modifications have been identified in RNA molecules. N6-methyladenosine (m6A) and 1-methyladenosine (m1A) are prevalent modifications occurring in various RNA species of mammals. Apart from the single methylation of adenosine (m6A and m1A), dual methylation modification occurring in the nucleobase of adenosine, such as N6,N6-dimethyladenosine (m6,6A), also has been reported to be present in RNA of mammals. Whether there are other forms of dual methylation modification occurring in the nucleobase of adenosine other than m6,6A remains elusive. Here, we reported the existence of a novel adenosine dual methylation modification, i.e. 1,N6-dimethyladenosine (m1,6A), in tRNAs of living organisms. We confirmed that m1,6A is located at position 58 of tRNAs and is prevalent in mammalian cells and tissues. The measured level of m1,6A ranged from 0.0049% to 0.047% in tRNAs. Furthermore, we demonstrated that TRMT6/61A could catalyze the formation of m1,6A in tRNAs and m1,6A could be demethylated by ALKBH3. Collectively, the discovery of m1,6A expands the diversity of RNA modifications and may elicit a new tRNA modification-mediated gene regulation pathway.

Comprehensive Profiling of Phosphomonoester Metabolites in Saccharomyces cerevisiae by the Chemical Isotope Labeling-LC-MS Method.

Phosphomonoesters are important biosynthetic and energy metabolism intermediates in microorganisms. A comprehensive analysis of phosphomonoester metabolites is of great significance for the understanding of their metabolic phosphorylation process and inner mechanism. In this study, we established a pair of isotope reagent d0/d5-2-diazomethyl-N-methyl-phenyl benzamide-labeling-based LC-MS method for the comprehensive analysis of phosphomonoester metabolites. By this method, the labeled phosphomonoester metabolites specifically produced characteristic isotope paired peaks with an m/z difference of 5.0314 in the MS1 spectra and a pair of diagnostic ions (m/z 320.0693/325.1077) in the MS2 spectra. Based on this, a diagnostic ion-based strategy was established for the rapid screening, identification, and relative quantification of phosphomonoester metabolites. Using this strategy, 42 phosphomonoester metabolites were highly accurately identified fromSaccharomyces cerevisiae (S. cerevisiae). Notably, two phosphomonoesters were first detected fromS. cerevisiae. The relative quantification results indicated that the contents of nine phosphomonoester metabolites including two intermediates (Ru5P and S7P) in the pentose phosphate pathway (PPP) were significantly different between lycopene-producible and wild-type S. cerevisiae. A further enzyme assay indicated that the activity of the PPP was closely related to the production of lycopene. Our findings provide new perspectives for the related mechanism study and valuable references for making informed microbial engineering decisions.

Single-Base Resolution Detection of N6-Methyladenosine in RNA by Adenosine Deamination Sequencing.

N6-Methyladenosine (m6A) is one of the most abundant and prevalent natural modifications occurring in diverse RNA species. m6A plays a wide range of roles in physiological and pathological processes. Revealing the functions of m6A relies on the faithful detection of individual m6A sites in RNA. However, developing a simple method for the single-base resolution detection of m6A is still a challenging task. Herein, we report an adenosine deamination sequencing (AD-seq) technique for the facile detection of m6A in RNA at single-base resolution. The AD-seq approach capitalizes on the selective deamination of adenosine, but not m6A, by the evolved tRNA adenosine deaminase (TadA) variant of TadA8e or the dimer protein of TadA-TadA8e. In AD-seq, adenosine is deaminated by TadA8e or TadA-TadA8e to form inosine, which pairs with cytidine and is read as guanosine in sequencing. m6A resists deamination due to the interference of the methyl group at the N6 position of adenosine. Thus, the m6A base pairs with thymine and is still read as adenosine in sequencing. The differential readouts from A and m6A in sequencing can achieve the single-base resolution detection of m6A in RNA. Application of the proposed AD-seq successfully identified individual m6A sites in Escherichia coli 23S rRNA. Taken together, the proposed AD-seq allows simple and cost-effective detection of m6A at single-base resolution in RNA, which provides a valuable tool to decipher the functions of m6A in RNA.

Engineered APOBEC3C Sequencing Enables Bisulfite-Free and Direct Detection of DNA Methylation at a Single-Base Resolution.

DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfite-free and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.

Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA.

Chemical modifications in DNA have profound influences on the structures and functions of DNA. Uracil, a naturally occurring DNA modification, can originate from the deamination of cytosine or arise from misincorporation of dUTP into DNA during DNA replication. Uracil in DNA will imperil genomic stability due to their potential in producing detrimental mutations. An in-depth understanding of the functions of uracil modification requires the accurate determination of its site as well as content in genomes. Herein, we characterized that a new member of the uracil-DNA glycosylase (UDG) family enzyme (UdgX-H109S) could selectively cleave both uracil-containing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Based on this unique property of UdgX-H109S, we developed an enzymatic cleavage-mediated extension stalling (ECES) method for the locus-specific detection and quantification of uracil in genomic DNA. In the ECES method, UdgX-H109S specifically recognizes and cleaves the N-glycosidic bond of uracil from dsDNA and generates an apurinic/apyrimidinic (AP) site, which could be broken by APE1 to form a one-nucleotide gap. The specific cleavage by UdgX-H109S is then evaluated and quantified by qPCR. With the developed ECES approach, we demonstrated that the level of uracil at position Chr4:50566961 in genomic DNA of breast cancer tissues was significantly decreased. Collectively, the ECES method has been proved to be accurate and reproducible in the locus-specific quantification of uracil in genomic DNA from biological and clinical samples.

DNA-Protein Cross-Linking Sequencing for Genome-Wide Mapping of Thymidine Glycol.

Thymidine glycol (Tg) is the most prevalent form of oxidatively induced pyrimidine lesions in DNA. Tg can arise from direct oxidation of thymidine in DNA. In addition, 5-methyl-2'-deoxycytidine (5-mdC) can be oxidized to 5-mdC glycol, and its subsequent deamination also yields Tg. However, Tg's distribution in the human genome remains unknown. Here, we presented a DNA-protein cross-linking sequencing (DPC-Seq) method for genome-wide mapping of Tg in human cells. Our approach capitalizes on the specificity of a bifunctional DNA glycosylase, i.e., NTHL1, for the covalent labeling, as well as DPC pulldown, SDS-PAGE fractionation, and membrane transfer for highly efficient and selective enrichment of Tg-bearing DNA. By employing DPC-Seq, we detected thousands of Tg sites in the human genome, where dual ablation of NTHL1 and NEIL1, the major DNA glycosylases responsible for Tg repair, led to pronounced increases in the number of Tg peaks. In addition, Tg is depleted in genomic regions associated with active transcription but enriched at nucleosome-binding sites, especially at heterochromatin sites marked with H3K9me2. Collectively, we developed a DPC-Seq method for highly efficient enrichment of Tg-containing DNA and for genome-wide mapping of Tg in human cells. Our work offers a robust tool for future functional studies of Tg in DNA, and we envision that the method can also be adapted for mapping other modified nucleosides in genomic DNA in the future.

Single-Base Resolution Detection of Adenosine-to-Inosine RNA Editing by Endonuclease-Mediated Sequencing.

RNA molecules contain diverse modifications that play crucial roles in a wide variety of biological processes. Adenosine-to-inosine (A-to-Ino) RNA editing is one of the most prevalent modifications among all types of RNA. Abnormal A-to-InoRNA editing has been demonstrated to be associated with many human diseases. Identification of A-to-Ino editing sites is indispensable to deciphering their biological roles. Herein, by employing the unique property of human endonuclease V (hEndoV), we proposed a hEndoV-mediated sequencing (hEndoV-seq) method for the single-base resolution detection of A-to-InoRNA editing sites. In this approach, the terminal 3'OH of RNA is first blocked by 3'-deoxyadenosine (3'-deoxy-A). Specific cleavage of Ino sites by hEndoV protein produces new terminal 3'OH, which can be identified by sequencing analysis, and therefore offers the site-specific detection of Ino in RNA. The principle of hEndoV-seq is straightforward and the analytical procedure is simple. No chemical reaction is involved in the sequencing library preparation. The whole procedure in hEndoV-seq is carried out under mild conditions and RNA is not prone to degradation. Taken together, the proposed hEndoV-seq method is capable of site-specific identification of A-to-Ino editing in RNA, which provides a valuable tool for elucidating the functions of A-to-Ino editing in RNA.

Identification of Inosine and 2'-O-Methylinosine Modifications in Yeast Messenger RNA by Liquid Chromatography-Tandem Mass Spectrometry Analysis.

The discovery of reversible modifications in messenger RNA (mRNA) opens new research directions in RNA modification-mediated epigenetic regulation. Yeast is an extensively used model organism in molecular biology. Systematic investigation and profiling of modifications in yeast mRNA would promote our understanding of the physiological regulation mechanisms in yeast. However, due to the high abundance of ribosomal RNA (rRNA) and transfer RNA (tRNA) in total RNA, isolation of low abundance of mRNA frequently suffers from the contamination of rRNA and tRNA, which will lead to the false-positive determination and inaccurate quantification of modifications in mRNA. Therefore, obtaining high-purity mRNA is critical for precise determination and accurate quantification of modifications in mRNA, especially for studies that focus on discovering new ones. Herein, we proposed a successive orthogonal isolation method by combining polyT-based purification and agarose gel electrophoresis purification for extracting high-purity mRNA. With the extracted high-purity yeast mRNA, we systemically explored the modifications in yeast mRNA by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The results showed that in addition to the previously reported eight kinds of modifications, two novel modifications of inosine (Ino) and 2'-O-methylinosine (Im) were identified to be prevalent in yeast mRNA. It is worth noting that Im was reported for the first time, to the best of our knowledge, to exist in living organisms in the three domains of life. Moreover, we observed that the levels of 10 kinds of modifications including Ino and Im in yeast mRNA exhibited dynamic change at different growth stages of yeast cells. Furthermore, Im in mRNA showed a significant decrease while in response to H2O2 treatment. These results indicated that the two newly identified modifications in yeast mRNA were involved in yeast cell growth and response to environmental stress. Taken together, we reported two new modifications of Ino and Im in yeast mRNA, which expends the diversity of RNA modifications in yeast and also suggests new regulators for modulating yeast physiological functions.

Ultrasensitive Determination of Sugar Phosphates in Trace Samples by Stable Isotope Chemical Labeling Combined with RPLC-MS.

Sugar phosphates are important metabolic intermediates in organisms and play a vital role in energy and central carbon metabolism. Profiling of sugar phosphates is of great significance but full of challenges due to their high structural similarity and low sensitivities in liquid chromatography (LC)-mass spectrometry (MS). In this study, we developed a novel stable isotope chemical labeling combined with the reversed-phase (RP)LC-MS method for ultrasensitive determination of sugar phosphates at the single-cell level. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-DMBA, sugar phosphate isomers can obtain better separation and identification, and the detection sensitivities of sugar phosphates increased by 3.5-147 folds. The obtained limits of detection of sugar phosphates ranged from 5 to 16 pg/mL. Using this method, we achieved ultrasensitive and accurate quantification of 12 sugar phosphates in different trace biological samples. Benefiting from the improved separation and detection sensitivity, we successfully quantified five sugar phosphates (d-glucose 1-phosphate, d-mannose 6-phosphate, d-fructose 6-phosphate, d-glucose 6-phosphate, and seduheptulose 7-phosphate) in a single protoplast of Arabidopsis thaliana.

Bisulfite-Free and Single-Base Resolution Detection of Epigenetic DNA Modification of 5-Methylcytosine by Methyltransferase-Directed Labeling with APOBEC3A Deamination Sequencing.

DNA methylation (5-methylcytosine, 5mC) is the most prevalent epigenetic modification that is predominantly found in CG dinucleotides in mammalian genomes. In-depth investigation of the functions of 5mC heavily relies on the quantitative measurement of 5mC at single-base resolution in genomes. Here, we proposed a methyltransferase-directed labeling with APOBEC3A (A3A) deamination sequencing (MLAD-seq) method for the single-base resolution and quantitative detection of 5mC in DNA. In MLAD-seq, a mutant of DNA methyltransferase, M.MpeI-N374K, is utilized to selectively transfer a carboxymethyl group to the 5 position of cytosine in the CG dinucleotide to form 5-carboxymethylcytosine (5camC) using carboxy-S-adenosyl-l-methionine (caSAM) as the cofactor. After A3A treatment, 5camC is resistant to the deamination and base pairs with guanine. Thus, the cytosines in CG sites are read as C in sequencing. On the contrary, the methyl group in 5mC inhibits its carboxymethylcytosine by M.MpeI-N374K and therefore is readily deaminated by A3A to produce thymine that pairs with adenine and is read as T in sequencing. The differential readouts from C and 5mC in the MLAD-seq enable the single-base resolution mapping of 5mC in CG sites in DNA. With the developed MLAD-seq method, we observed the hypermethylation in the promoter region of retinoic acid receptor ß (RARB) gene from human nonsmall cell lung tumor tissue. Compared to harsh reaction conditions in bisulfite sequencing that could lead to significant degradation of DNA, the whole procedure of MLAD-seq is carried out under mild conditions, which will avoid DNA damage. Thus, MLAD-seq is more suitable in the scenario where only limited input DNA is available. Taken together, the MLAD-seq offers a valuable tool for bisulfite-free, single-base resolution and quantitative detection of 5mC in limited DNA.

Photoactive G-Quadruplex Ligand Identifies Multiple G-Quadruplex-Related Proteins with Extensive Sequence Tolerance in the Cellular Environment.

G-Quadruplex (G4) is a noncanonical nucleic acid secondary structure with multiple biofunctions. Identifying G4-related proteins (G4RPs) is important for understanding the roles of G4 in biology. Current methods to identify G4RPs include discovery from specific biological processes or in vitro pull-down assays with specific G4 sequences. Here, we report an in vivo strategy used to identify G4RPs with extensive sequence tolerance based on G4 ligand-mediated cross-linking. Applying this method, we identified 114 and 281 G4RPs in SV589 and MM231 cells, respectively. The results successfully overlapped with all the pull-down assay literature. Through the electrophoretic mobility shift assay (EMSA), we identified some new G4-binding proteins. Moreover, enhanced cross-linking and immunoprecipitation (eCLIP) confirmed that one newly identified G4-binding protein, SERBP1, interacts with G4 in the cellular environment. The method we developed provides a new strategy for identifying proteins that interact with nucleic secondary structures in cells and benefit the study of their biological roles.

Sensitive and Simultaneous Determination of Uridine Thiolation and Hydroxylation Modifications in Eukaryotic RNA by Derivatization Coupled with Mass Spectrometry Analysis.

The discovery of dynamic and reversible modifications in RNA expands their functional repertoires. Now, RNA modifications have been viewed as new regulators involved in a variety of biological processes. Among these modifications, thiolation is one kind of special modification in RNA. Several thiouridines have been identified to be present in RNA, and they are essential in the natural growth and metabolism of cells. However, detection of these thiouridines generally is challenging, and few studies could offer the quantitative levels of uridine modifications in RNA, which limits the in-depth elucidation of their functions. Herein, we developed a chemical derivatization in combination with mass spectrometry analysis for the sensitive and simultaneous determination of uridine thiolation and hydroxylation modifications in eukaryotic RNA. The chemical derivatization strategy enables the addition of easily ionizable groups to the uridine thiolation and hydroxylation modifications, leading up to a 339-fold increase in detection sensitivities of these modifications by mass spectrometry analysis. The limits of detection of these uridine modifications can be down to 17 amol. With the established method, we discovered and confirmed that a new modification of 5-hydroxyuridine (ho5U) was widely present in small RNAs of mammalian cells, expanding the diversity of RNA modifications. The developed method shows superior capability in determining low-abundance RNA modifications and may promote identifying new modifications in RNA, which should be valuable in uncovering the unknown functions of RNA modifications.

Chemical Tagging Assisted Mass Spectrometry Analysis Enables Sensitive Determination of Phosphorylated Compounds in a Single Cell.

Polar phosphorylated metabolites are involved in a variety of biological processes and play vital roles in energetic metabolism, cofactor regeneration, and nucleic acid synthesis. However, it is often challenging to interrogate polar phosphorylated metabolites and compounds from biological samples. Liquid chromatography-mass spectrometry (LC/MS) now plays a central role in metabolomic studies. However, LC/MS-based approaches have been hampered by the issues of the low ionization efficiencies, low in vivo concentrations, and less chemical stability of polar phosphorylated metabolites. In this work, we synthesized paired reagents of light and heavy isotopomers, 2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (DMPI) and d3-(2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (d3-DMPI). The paired reagents of DMPI and d3-DMPI carry diazo groups that can efficiently and selectively react with the phosphate group on polar phosphorylated metabolites under mild conditions. As a proof of concept, we found that the transfer of the indole heterocycle group from DMPI/d3-DMPI to ribonucleotides led to the significant increase of ionization efficiencies of ribonucleotides during LC/MS analysis. The detection sensitivities of these ribonucleotides increased by 25-1137-fold upon DMPI tagging with the limits of detection (LODs) being between 7 and 150 amol. With the developed method, we achieved the determination of all the 12 ribonucleotides from a single mammalian cell and from a single stamen of Arabidopsis thaliana. The method provides a valuable tool to investigate the dynamic changes of polar phosphorylated metabolites in a single cell under particular conditions.

N 6-Hydroxymethyladenine: a hydroxylation derivative of N6-methyladenine in genomic DNA of mammals.

In addition to DNA cytosine methylation (5-methyl-2'-deoxycytidine, m5dC), DNA adenine methylation (N6-methyl-2'-deoxyadenosine, m6dA) is another DNA modification that has been discovered in eukaryotes. Recent studies demonstrated that the content and distribution of m6dA in genomic DNA of vertebrates and mammals exhibit dynamic regulation, indicating m6dA may function as a potential epigenetic mark in DNA of eukaryotes besides m5dC. Whether m6dA undergoes the further oxidation in a similar way to m5dC remains elusive. Here, we reported the existence of a new DNA modification, N6-hydroxymethyl-2'-deoxyadenosine (hm6dA), in genomic DNA of mammalian cells and tissues. We found that hm6dA can be formed from the hydroxylation of m6dA by the Fe2+- and 2-oxoglutarate-dependent ALKBH1 protein in genomic DNA of mammals. In addition, the content of hm6dA exhibited significant increase in lung carcinoma tissues. The increased expression of ALKBH1 in lung carcinoma tissues may contribute to the increase of hm6dA in DNA. Taken together, our study reported the existence and formation of hm6dA in genomic DNA of mammals.

Quantitative Analysis of Oncometabolite 2-Hydroxyglutarate.

Gain-of-function mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) were demonstrated to induce the production and accumulation of oncometabolite 2-hydroxyglutarate (2HG). 2HG is a potent competitor of α-ketoglutarate (α-KG) and can inhibit multiple α-KG-dependent dioxygenases that are critical for regulating the metabolic and epigenetic state of cells. The accumulation of 2HG contributes to elevated risk of malignant tumors. 2HG carries an asymmetric carbon atom in its carbon backbone and therefore occurs in two enantiomers, D-2-hydroxyglutarate (D-2HG) and L-2-hydroxyglutarate (L-2HG). Each enantiomer is produced and metabolized in independent biochemical pathway and catalyzed by different enzymes. The accurate diagnosis of 2HG-related diseases relies on determining the configuration of the two enantiomers. Quantitative methods for analysis of D-2HG and L-2HG have been well developed. These analytical strategies mainly include the use of chiral chromatography medium to facilitate chromatographic separation of enantiomers prior to spectroscopy or mass spectrometry analysis and the use of chiral derivatization reagents to convert the enantiomers to diastereomers with differential physical and chemical properties that can improve their chromatographic separation. Here, we summarize and discuss these established methods for analysis of total 2HG as well as the determination of the enantiomers of D-2HG and L-2HG.