An on-line SPE-LC-MS/MS method for quantification of nucleobases and nucleosides present in biological fluids.

Solid phase extraction (SPE) and liquid chromatographic (LC) separation of nucleobases and nucleosides are challenging due to the high hydrophilicity of these compounds. Herein we report a novel on-line SPE-LC-MS/MS method for their quantification after pre-column derivatization with chloroacetaldehyde (CAA). The method proposed is selective and sensitive with limits of detection at the nano-molar level. Analysis of urine and saliva samples by using this method is demonstrated. Adenine, guanine, cytosine, adenosine, guanosine, and cytidine were found in the range from 0.19 (guanosine) to 1.83 µM (cytidine) in urine and from 0.015 (guanosine) to 0.79 µM (adenine) in saliva. Interestingly, methylation of cytidine was found to be significantly different in urine from that in saliva. While 5-hydroxymethylcytidine was detected at a very low level (<0.05 µM) in saliva, it was found to be the most prominent methylated cytidine in urine at a high level of 3.33 µM. Since on-line SPE is deployed, the proposed LC-MS/MS quantitative assay is convenient to carry out and offers good assay accuracy and repeatability.

A comprehensive evaluation of a polymeric zwitterionic hydrophilic monolith for nucleotide separation.

Rapid and effective separation of nucleotides (NTs) and their derivatives is crucial for studying their physiological functions. In this work, we comprehensively evaluated the separation ability of a zwitterionic hydrophilic monolith, i.e., poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammonium betaine-co-N,N'-methylenebisacrylamide) (poly(SPP-co-MBA)) for NTs analysis, including its selectivity, chemical stability under extremely basic condition and compatibility with hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (HILIC-MS). The poly(SPP-co-MBA) monolith exhibited excellent chemical stability, as evidenced by the low relative standard deviation of retention time (0.16-1.05%) after 4000 consecutive injections over one month under strong alkaline elution condition (pH 10). After optimizing the separation conditions, including buffer pH and concentration, organic solvent content and column temperature, four nucleoside triphosphates, five nucleoside diphosphates and five nucleoside monophosphates were baseline separated within 7 min. Additionally, the mixtures containing one nucleoside and its corresponding mono-, di-, and triphosphates were baseline separated within only 3 min, respectively. It is good HILIC-MS compatibility was also confirmed by the satisfactory peak shape and high response of nine NTs. Overall, the proposed poly(SPP-co-MBA) monolith exhibited good mechanical stability and compatibility of HILIC-MS, making it a promising technique for NTs analysis.

Automated Identification of Modified Nucleosides during HRAM-LC-MS/MS using a Metabolomics ID Workflow with Neutral Loss Detection.

The role of post-transcriptional modification in biological processes has been an ongoing field of study for several decades. Improvements in liquid chromatography platforms and mass spectrometry instrumentation have resulted in the enhanced identification, characterization, and quantification of modified nucleosides in biological systems. One consequence of the rapid technological improvements in the analytical acquisition of modified nucleosides has been a dearth of robust data processing workflows for analyzing more than a handful of samples at a time. To improve the utility of LC-MS/MS for batch analyses of modified nucleosides, a workflow for automated nucleoside identification has been developed. We adapted the Thermo Fisher Scientific metabolomics identification software package, Compound Discoverer, to accurately identify modified nucleosides from batch LC-MS/MS acquisitions. Three points of identification are used: accurate mass from a monoisotopic mass list, spectral matching from a spectral library, and neutral loss identification. This workflow was applied to a batch (n = 24) of urinary nucleosides, resulting in the accurate identification and relative quantification of 16 known nucleosides in less than 1 h.

Comparing the separation performance of poly(ethyleneimine) embedded butyric and octanoic acid based chromatographic stationary phases.

Poly(ethyleneimine) (PEI) modified silica spheres were used to graft butyric acid and octanoic acid onto their surfaces, forming two stationary phases named Sil-PEI-BAD and Sil-PEI-CAD, respectively. Characterized methods including fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA) and thermogravimetric analysis (TGA) were utilized to determine the successful synthesis of these two stationary phase materials. The chromatographic performance of these two stationary phases was analyzed with hydrophobic and hydrophilic compounds as analytes. Compared with Sil-PEI-CAD column, Sil-PEI-BAD column was more effective in separating hydrophilic compounds including nucleosides, alkaloids and vitamins. Hydrophobic substances including polycyclic aromatic hydrocarbons (PAHs) and alkylbenzenes obtained excellent separation results on Sil-PEI-CAD column than Sil-PEI-BAD column. Additionally, according to the separation of phenols, Sil-PEI-CAD column can be used in HILIC/RPLC mixed-mode. The results showed that the properties and retention mechanisms of the prepared stationary phases depended on the length of the alkyl chains bonded on the silica surface.

Targeted quantitative metabolomics with a linear mixed-effect model for analysis of urinary nucleosides and deoxynucleosides from bladder cancer patients before and after tumor resection.

In the present study, we developed and validated a fast, simple, and sensitive quantitative method for the simultaneous determination of eleven nucleosides and deoxynucleosides from urine samples. The analyses were performed with the use of liquid chromatography coupled with triple quadrupole mass spectrometry. The sample pretreatment procedure was limited to centrifugation, vortex mixing of urine samples with a methanol/water solution (1:1, v/v), evaporation and dissolution steps. The analysis lasted 20 min and was performed in dynamic multiple reaction monitoring mode (dMRM) in positive polarity. Process validation was conducted to determine the linearity, precision, accuracy, limit of quantification, stability, recovery and matrix effect. All validation procedures were carried out in accordance with current FDA and EMA regulations. The validated method was applied for the analysis of 133 urine samples derived from bladder cancer patients before tumor resection and 24 h, 2 weeks, and 3, 6, 9, and 12 months after the surgery. The obtained data sets were analyzed using a linear mixed-effect model. The analysis revealed that concentration level of 2-methylthioadenosine was decreased, while for inosine, it was increased 24 h after tumor resection in comparison to the preoperative state. The presented quantitative longitudinal study of urine nucleosides and deoxynucleosides before and up to 12 months after bladder tumor resection brings additional prospective insight into the metabolite excretion pattern in bladder cancer disease. Moreover, incurred sample reanalysis was performed proving the robustness and repeatability of the developed targeted method.

Multiplexed LC-MS/MS quantification of salivary RNA modifications in periodontitis.

OBJECTIVE: To analyse the salivary epitranscriptomic profiles as periodontitis biomarkers using multiplexed mass spectrometry (MS). BACKGROUND: The field of epitranscriptomics, which relates to RNA chemical modifications, opens new perspectives in the discovery of diagnostic biomarkers, especially in periodontitis. Recently, the modified ribonucleoside N6-methyladenosine (m6A) was revealed as a crucial player in the etiopathogenesis of periodontitis. However, no epitranscriptomic biomarker has been identified in saliva to date. MATERIALS AND METHODS: Twenty-four saliva samples were collected from periodontitis patients (n = 16) and from control subjects (n = 8). Periodontitis patients were stratified according to stage and grade. Salivary nucleosides were directly extracted and, in parallel, salivary RNA was digested into its constituent nucleosides. Nucleoside samples were then quantified by multiplexed MS. RESULTS: Twenty-seven free nucleosides were detected and an overlapping set of 12 nucleotides were detected in digested RNA. Among the free nucleosides, cytidine and three other modified nucleosides (inosine, queuosine and m6Am) were significantly altered in periodontitis patients. In digested RNA, only uridine was significantly higher in periodontitis patients. Importantly there was no correlation between free salivary nucleoside levels and the levels of those same nucleotides in digested salivary RNA, except for cytidine, m5C and uridine. This statement implies that the two detection methods are complementary. CONCLUSION: The high specificity and sensitivity of MS allowed the detection and quantification of multiple nucleosides from RNA and free nucleosides in saliva. Some ribonucleosides appear to be promising biomarkers of periodontitis. Our analytic pipeline opens new perspectives for diagnostic periodontitis biomarkers.

Metabolomics in Plant Research-From Ecometabolomics to Metabolotyping.

In this Special Issue, a state-of-the-art review of the current knowledge of sample preparation and LC-MS techniques for the analyses of nucleosides and nucleotides in plants was published [...].

Zwitterionic polymer-terminated porous silica stationary phases for highly selective separation in hydrophilic interaction chromatography.

We described two novel zwitterionic polymer-terminated porous silica stationary phases containing the same pyridinium cation and anions of different side chains (carboxylate and phosphonate groups) for use in hydrophilic interaction liquid chromatography (HILIC). These two novel columns were prepared by polymerizing 4-vinylpyridine and grafting it onto a silica surface, followed by quaternization reaction with 3-bromopropionic acid (Sil-VPC24) and (3-bromopropyl) phosphonic acid (Sil-VPP24), which possess positively charged pyridinium groups, and negatively charged carboxylate and phosphonate groups, respectively. The products obtained were verified through relevant characterization techniques such as elemental analysis, Fourier-transform infrared spectroscopy, thermogravimetric analysis, Zeta potential analysis, and Brunauer-Emmett-Teller analysis. The retention properties and mechanisms of different types of compounds (neutral, cationic, and anionic) on the two zwitterionic-modified silica stationary phases were studied by varying the buffer salt concentration and pH of the eluent. The separation of phenol and aromatic acids, disubstituted benzene isomers, sulfonamide drugs, as well as nucleosides/nucleobases were investigated on the two packed novel columns and a commercial zwitterionic column in identical HILIC mode, ensuring a thorough comparison between both novel columns and with a commercial standard. The results illustrated that various compounds could be separated up to various efficiencies based on the mechanism of hydrophilic interaction-based retention between the solutes and the two zwitterionic polymer stationary phases. The Sil-VPP24 column demonstrated the best separation performance out of the three, as well as flexible selectivity and excellent resolution. Both novel columns exhibited excellent stability and chromatographic repeatability for the separation of seven nucleosides and bases.

Nucleos'ID: A New Search Engine Enabling the Untargeted Identification of RNA Post-transcriptional Modifications from Tandem Mass Spectrometry Analyses of Nucleosides.

As RNA post-transcriptional modifications are of growing interest, several methods were developed for their characterization. One of them established for their identification, at the nucleosidic level, is the hyphenation of separation methods, such as liquid chromatography or capillary electrophoresis, to tandem mass spectrometry. However, to our knowledge, no software is yet available for the untargeted identification of RNA post-transcriptional modifications from MS/MS data-dependent acquisitions. Thus, very long and tedious manual data interpretations are required. To meet the need of easier and faster data interpretation, a new user-friendly search engine, called Nucleos'ID, was developed for CE-MS/MS and LC-MS/MS users. Performances of this new software were evaluated on CE-MS/MS data from nucleoside analyses of already well-described Saccharomyces cerevisiae transfer RNA and Bos taurus total tRNA extract. All samples showed great true positive, true negative, and false discovery rates considering the database size containing all modified and unmodified nucleosides referenced in the literature. The true positive and true negative rates obtained were above 0.94, while the false discovery rates were between 0.09 and 0.17. To increase the level of sample complexity, untargeted identification of several RNA modifications from Pseudomonas aeruginosa 70S ribosome was achieved by the Nucleos'ID search following CE-MS/MS analysis.

Preparation of three regioisomeric ionic liquid stationary phases and investigation of their retention behavior.

The structural properties of ionic liquid stationary phases have a considerable effect on their separation selectivity. However, the difference of the chromatographic retention behavior of different regioisomeric ionic liquid stationary phases has rarely been investigated. In this study, three regioisomeric ionic liquid silane reagents were prepared by photoinitiated ene-click chemistry and bonded to silica by one-pot method to fabricate three new stationary phases (Sil-C2Im-C8, Sil-C6Im-C4, and Sil-C9Im-C1). All three stationary phases showed promising retention repeatability and efficiency. The retention behavior of the three stationary phases was investigated under various chromatographic conditions. The retention mechanism was further investigated by the linear energy solvation relationship and Van't Hoff plots. The stationary phases exhibited mixed-mode retention mechanisms. The π-π, hydrogen bonding, ion-exchange, and hydrophilic interactions with analytes were the weakest when the imidazole ions were embedded in the innermost part of the alkyl chains, while the interactions were the strongest when the imidazole ions were embedded in the middle of the alkyl chains. The three stationary phases provided great but different separation performances towards nucleosides, nucleobases, aromatic acids, alkyl benzenes, and polycyclic aromatic hydrocarbons due to the influence of imidazole ion position.

Quantitative multicomponent analysis of a single marker-based simultaneous determination and quality assessment of nucleoside analogs from Qi Zhi capsules.

Qi Zhi capsule (QZC) is approved by the State Drug Administration of China. The QZC consists of nine crude drugs, including astragalus, leeches, ground beetles, curcuma zedoary, hawthorn, semen cassiae, rhizoma sparganii, polygonum multiflorum, and peach kernel, of which leeches and ground beetles are Traditional Chinese Medicine of animal origin. Nucleosides are animal substances with pharmacological effects that are easy to extract and quantify. Different nucleoside analogs in distinct animal-based formulations can be used to characterize animal-based medicines. However, the quality control of a single indicator does not reflect the overall quality of Chinese medicine. Here, we developed a method to simultaneously determine the nucleoside analogs uracil, xanthine, hypoxanthine, uridine, guanine, and uric acid in QZCs using high-performance liquid chromatography. Hypoxanthine was used as an internal reference to determine relative correction factors for the other five components. The six components were determined in ten different batches of QZCs. There was no significant difference between the quantitative multicomponent analysis of a single marker and the external standard method. The relative standard deviation of total nucleosides analogs of 10 batches of samples was 7%. This method can be applied to simultaneously determine multiple active components in QZCs and other nucleoside analog drugs, enabling multi-indicator quality control.

A review of traditional and current processing methods used to decrease the toxicity of the rhizome of Pinellia ternata in traditional Chinese medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Pinellia ternata (Thunb.) Breit, called Pinelliae Rhizoma (PR) and Banxia in Chinese, is a well-known traditional Chinese medicine (TCM) with the functions of "removing dampness-phlegm" and "downbear counterflow and check vomiting". PR has potential toxic effects that can be detoxified by Fuzhi processing (repeated processing using one or multiple adjuvants) with specific adjuvants. AIM OF THE STUDY: This paper aims to provide a summary of traditional and current processing methods used to detoxify PR. MATERIALS AND METHODS: The available references of the processing methods of PR from the classic books of Materia Medica, literature, online databases and masters or doctoral theses are collected and summarized. We also discussed the possible processing mechanisms of how we can achieve a safer and effective application of PR via these processing methods. RESULTS: PR cannot be administered orally before processing. PR contains nucleoside alkaloids, cerebrosides, fatty acids, lectin, polysaccharides, and calcium oxalate crystals. To date, although the active substances of PR are still unclear, the toxic components are almost completely clarified as needle-like calcium oxalate crystals (NCOCs) and lectin proteins. Furthermore, the toxic effects of PR include causing death in animals, inflammation, conjunctival irritation, pregnancy toxicity, teratogenicity, visceral toxicity, aphonia and vomiting. From ancient times to now, Fuzhi methods have remained the predominant method for PR processing, and the main adjuvants used are ginger juice, alum, licorice and lime. In addition, detoxification mechanisms are related to removing or damaging the NCOC and lectin in PR based on processing with adjuvants. Currently, Fuzhi processing has been greatly improved, and novel processing technologies with novel adjuvants have been used for PR processing. However, there are still some flaws in PR processing, which should be urgently solved in the future, and clarifying the characteristic bioactive compounds in PR corresponding to its function or effects is the most important step for PR processing. CONCLUSION: Our present paper reviewed the previous literature regarding all aspects of the processing of PR, and this paper will be helpful for achieving a safer and effective application of PR and its processed products and will also be beneficial for the further optimization of processing technology and clinical medication safety of PR.

Quantitative analysis of nutrients for nucleosides, nucleobases, and amino acids hidden behind five distinct regions-derived Poria cocos using ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry.

Poria cocos is an edible fungus used as a health product and traditional Chinese medicinal preparation. Nevertheless, little is known about its nutrients. In this study, ultra-high performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry was conducted to quantify nucleosides, nucleobases, and amino acids in 32 batches of Poria cocos samples collected from Anhui, Sichuan, Hubei, Hunan, and Guizhou. Subsequently, the linearity, precision, repeatability, stability, and recovery of our methods were validated. Samples from different regions were clearly separated by partial least squares discriminant analysis and cluster analysis. Our results suggested that Poria cocos samples from different geographical environments differed in nucleosides, nucleobases, and amino acids. The plot of variable importance for projection disclosed differential compositions of L-Leucine, Uridine, L-Asparagine, L-Glutamine, L-phenylalanine, L-Ornithine monohydrochloride, L-Hydroxyproline, Taurine, and Inosine in Poria cocos from five regions. We found the highest content of total analytes, total amino acids, and total non-essential amino acids in Poria cocos from Anhui, total essential amino acids in the Sichuan samples, and total nucleosides in the Hunan samples. Overall, we determined the content of Poria cocos-derived nucleosides, nucleobases, and amino acids, providing the foothold for further chemical mining and use of Poria cocos.

Optimization of nucleotides dephosphorylation for RNA structural characterization by tandem mass spectrometry hyphenated with separation methods.

As part of RNA characterization, the identification of post-transcriptional modifications can be performed using hyphenation of separation methods with mass spectrometry. To identify RNA modifications with those methods, a first total digestion followed by a dephosphorylation step are usually required to reduce RNA to nucleosides. Even though effective digestion and dephosphorylation are essential to avoid further complications in analysis and data interpretation, to our knowledge, no standard protocol is yet referenced in the literature. Therefore, the aim of this work is to optimize the dephosphorylation step using a total extract of transfer RNA (tRNA)1 from B. taurus as a model and to determine and fix two protocols, leading to complete dephosphorylation, based on time and bacterial alkaline phosphatase (BAP)2 consumptions. Capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) was used to estimate the dephosphorylation efficiency of both protocols on many canonical and modified nucleotides. For a timesaving protocol, we established that full dephosphorylation was obtained after a 4-hour incubation at 37 °C with 7.5 U of BAP for 1 µg of tRNA. And for a BAP-saving protocol, we established that full dephosphorylation was obtained 3.0 U of BAP after an overnight incubation at 37 °C. Both protocols are suitable for quantitative analyses as no loss of analytes is expected. Moreover, they can be widely used for all other RNA classes, including messenger RNA or ribosomal RNA.

Protocol to analyze and quantify protein-methylated RNA interactions in mammalian cells with a combination of RNA immunoprecipitation and nucleoside mass spectrometry.

Cellular RNAs are modified by both physiological factors and exogenous agents, such as methyl methanesulfonate (MMS). However, techniques for analyzing how proteins may interact with these modified RNAs are limited. Here, we provide a protocol combining RNA immunoprecipitation (RIP) with mass spectrometry (MS) to analyze the methylation state of the RNAs bound by Flag-tagged proteins in mammalian cells. The approach is highly quantitative and can simultaneously detect several methylated nucleosides in a single experiment. For complete details on the use and execution of this protocol, please refer to Tsao et al. (2021).

An Overview of Pretreatment and Analysis of Nucleotides in Different Samples (Update since 2010).

Nucleotides, which are important low-molecular-weight compounds present in organisms, are precursors of nucleic acids and participate in various regulatory and metabolic functions. Sensitive and valid methods for monitoring and determining nucleotides and nucleosides in different samples are urgently required. Due to the presence of numerous endogenous interferences in complex matrices and the high polarity of the molecules of the phosphate moiety, the determination of nucleotide content is challenging. This review summarizes the pretreatment and analysis methods of nucleotides in different samples. Advanced pretreatment methods, including different microextraction methods, solid-phase extraction based on novel materials, QuEChERS, are clearly displayed, and continuous progress which has been made in LC, LC-MS/MS and capillary electrophoresis methods are discussed. Moreover, the strengths and weaknesses of different methods are discussed and compared. Highlight:Advanced pretreatment and detection methods of nucleotides were critically reviewed.Microextraction technology was one of the trends of nucleotides pretreatment in the future.Applications of novel materials and supercritical fluid were highlighted.The evolution and advance of HRMS analyzers were in detailed.

Quality Evaluation of Taxilli Herba from Different Hosts Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.

Protocol for preparation and measurement of intracellular and extracellular modified RNA using liquid chromatography-mass spectrometry.

About 150 modifications have been identified in RNA species. Besides their regulatory roles in the intracellular gene expression, abundant modified RNA nucleosides are catabolized from RNA and released into extracellular fluids, which can impact extracellular signaling as ligands for receptors. Here, we describe a protocol to prepare samples from biological specimens, including cultured cells, extracellular fluid, and tissues, to measure both intracellular and extracellular RNA modifications using mass spectrometry. For complete details on the use and execution of this protocol, please refer to Ogawa et al. (2021).

Soybean (Glycine max L Merr) host-plant defenses and resistance to the two-spotted spider mite (Tetranychus urticae Koch).

In southern Ontario, Canada, the two-spotted spider mite (Tetranychus urticae) is an emerging pest of soybean (Glycine max) due to the increasing incidence of warmer, drier weather conditions. One key strategy to manage soybean pests is breeding resistant cultivars. Resistance to pathogens and herbivores in soybean has been associated with isoflavonoid phytoalexins, a group of specialized metabolites commonly associated with root, leaf and seed tissues. A survey of 18 Ontario soybean cultivars for spider mite resistance included evaluations of antibiosis and tolerance in relation to isoflavonoid and other metabolites detected in the leaves. Ten-day and 4-week trials beginning with early growth stage plants were used to compare survival, growth, fecundity as well as damage to leaves. Two-spotted spider mite (TSSM) counts were correlated with HPLC measurements of isoflavonoid concentration in the leaves and global metabolite profiling by high resolution LC-MS to identify other metabolites unique to the most resistant (R) and susceptible (S) cultivars. Within 10 days, no significant difference (P>0.05) in resistance to TSSM was determined between cultivars, but after 4 weeks, one cultivar, OAC Avatar, was revealed to have the lowest number of adult TSSMs and their eggs. Other cultivars showing partial resistance included OAC Wallace and OAC Lakeview, while Pagoda was the most tolerant to TSSM feeding. A low, positive correlation between isoflavonoid concentrations and TSSM counts and feeding damage indicated these compounds alone do not explain the range of resistance or tolerance observed. In contrast, other metabolite features were significantly different (P<0.05) in R versus S cultivars. In the presence of TSSM, the R cultivars had significantly greater (P<0.05) concentrations of the free amino acids Trp, Val, Thr, Glu, Asp and His relative to S cultivars. Furthermore, the R cultivar metabolites detected are viable targets for more in-depth analysis of their potential roles in TSSM defense.

Quantitative analysis of m6A RNA modification by LC-MS.

N6-adenosine methylation (m6A) of messenger RNA (mRNA) plays key regulatory roles in gene expression. Accurate measurement of m6A levels is thus critical to understand its dynamic changes in various biological settings. Here, we provide a protocol to quantitate the levels of adenosine and m6A in cellular mRNAs. Using nuclease and phosphatase, we digest mRNA into nucleosides, which are subsequently quantified using liquid chromatography mass spectrometry. For complete details on the use and execution of this protocol, please refer to Cho et al. (2021).