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).

End-labeling-based electrochemical strategy for detection of adenine methylation in nucleic acid by differential pulse voltammetry.

A promising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on a gold nanoparticle-modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase-labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein.

DNA N6-methyladenine increased in human esophageal squamous cell carcinoma.

Esophageal cancer is one of the most common malignancies worldwide. DNA N6-methyladenine (6mA) has been well-studied in prokaryotes, while the distribution and biological functions of DNA 6mA in eukaryotic cells remain to be elucidated. Recently, DNA 6mA epigenetic modification was found in human gastric and liver cancers. To explore the status of DNA 6mA epigenetic modification in esophageal cancer, 101 cases of human esophageal squamous cell carcinoma (ESCC) and matched adjacent normal tissue samples were analyzed by dot blot assay. The levels of genomic DNA 6mA were significantly higher in ESCC tissue samples than in matched adjacent normal tissue samples (P<0.001). Increased DNA 6mA was associated with poor tumor differentiation (P<0.05), while no association was found between 6mA modification and gender, age, tumor size, TNM stage, lymph node metastasis, smoking, alcohol intake, or family history (all P>0.05). In conclusion, DNA 6mA epigenetic modification increased in human ESCC and may serve as a prognostic marker.

Effects of Extraction Temperature on Efficacy of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), Aqueous Extract against Oxidative Stress.

This study was designed to understand the effect of extraction temperature, i.e., room temperature (GLRT), 50°C (GL50), 100°C (hot water; GL100), and 200°C (GL200) on antioxidant and biological activity of G. lucidum. The % yield obtained was 5.3%, 7.6%, 10.7%, and 13.2% at various extraction temperatures; room temperature, 50°C, 100°C and 200°C, respectively. Similarly, phenolic content (51.6, 57.9, 82.9, and 93.1 mg/g extract) and flavonoid content (18.8, 23.2, 34.3, and 36.3 mg/g extract) were observed to be increased with rise in extraction temperature. However, extraction temperature resulted in loss of antioxidant activities above 100°C as evident by chemical assays such as DPPH, FRAP, ABTS, and TRP conducted on extracts. In contrast, three bioactive compounds, i.e., adenine (3.26, 3.48, 2.16, and 1.45 mg/g extract), uracil (3.99, 3.21, 2.51, and 1.47 mg/g extract), and adenosine (5.92, 5.62, 2.22 and 0.7 mg/g extract), quantified by high performance thin layer chromatography showed decrease in their content with increasing extraction temperature. Extract prepared at room temperature and 50°C prevented loss of cell viability and generation of reactive oxygen species resulted after hydrogen peroxide exposure; however, cytoprotective efficacy was not significant at 100°C and 200°C The order of cytoprotective effects observed by these extract were in the following order: room temperature ≥ 50°C > 100°C > 200°C. Overall, the optimal temperature conditions for the efficient extraction of G. lucidum with water retaining bioactive compounds and biological activity was found to be below 100°C.

Ground and Excited States of Gas-Phase DNA Nucleobase Cation-Radicals. A UV-vis Photodisociation Action Spectroscopy and Computational Study of Adenine and 9-Methyladenine.

Cation radicals of adenine (A•+) and 9-methyladenine (MA•+) were generated in the gas phase by collision-induced intramolecular electron transfer in copper-terpyridine-nucleobase ternary complexes and characterized by collision-induced dissociation (CID) mass spectra and UV-vis photodissociation action spectroscopy in the 210-700 nm wavelength region. The action spectra of both A•+ and MA•+ displayed characteristic absorption bands in the near-UV and visible regions. Another tautomer of A•+ was generated as a minor product by multistep CID of protonated 9-(2-bromoethyl)adenine. Structure analysis by density functional theory and coupled-clusters ab initio calculations pointed to the canonical 9-H-tautomer Ad1•+ as the global energy minimum of adenine cation radicals. The canonical tautomer MA1•+ was also calculated to be a low-energy structure among methyladenine cation radicals. However, two new noncanonical tautomers were found to be energetically comparable to MA1•+. Vibronic absorption spectra were calculated for several tautomers of A•+ and MA•+ and benchmarked on equation-of-motion coupled-clusters excited-state calculations. Analysis of the vibronic absorption spectra of A•+ tautomers pointed to the canonical tautomer Ad1•+ as providing the best match with the action spectrum. Likewise, the canonical tautomer MA1•+ was the unequivocal best match for the MA•+ ion generated in the gas phase. According to potential-energy mapping, MA1•+ was separated from energetically favorable noncanonical cation radicals by a high-energy barrier that was calculated to be above the dissociation threshold for loss of a methyl hydrogen atom, thus preventing isomerization. Structures and energetics of all four DNA nucleobase cation radicals are compared and discussed.

Upper urinary tract stone compositions: the role of age and gender

ABSTRACT Objective: To analyze the compositions of upper urinary tract stones and investigate their distributions in different gender and age groups. Materials and Methods: Patients diagnosed with upper urinary tract stone disease between December 2014 and March 2018 were retrospectively reviewed. Patient's age, gender, BMI, comorbidities, stone event characteristics, and compositions were collected, and proportions of stone components in different gender and age groups were analyzed. Results: A total of 1532 stone analyses were performed (992 from males and 540 from females). The mean age was younger in males (p <0.001). Males included more cases with larger BMI, hyperuricemia, and obesity, while females had more urinary tract infections. Multiple components were present in 61.8% of stones. Calcium oxalate (CaOx) (67.0%) was the most common component, followed by uric acid (UA) (11.8%), infection stone (11.4%), calcium phosphate (CaP) (8.0%), cystine (1.1%), brushite (0.4%), and 2, 8-dihydroxyadenine (0.2%). Men contributed with more CaOx stones than women at age 30-49 years (all p <0.01) and more UA stones at 30-59 years (all p <0.05). Women contributed with more infection stones than men in age groups 30-49 and 60-69 years (all p <0.05), and more CaP stones at 30-49 years. The prevalence peak was 50-59 years in men and 60-69 years in women. Both genders had the lowest prevalence in adolescence. Prevalence of UA stones increased while that of infection stones decreased with aging in both genders. Conclusions: Age and sex had a strong association with distribution of stone compositions in this Chinese cohort.

Upper urinary tract stone compositions: the role of age and gender.

OBJECTIVE: To analyze the compositions of upper urinary tract stones and investigate their distributions in different gender and age groups. MATERIALS AND METHODS: Patients diagnosed with upper urinary tract stone disease between December 2014 and March 2018 were retrospectively reviewed. Patient's age, gender, BMI, comorbidities, stone event characteristics, and compositions were collected, and proportions of stone components in different gender and age groups were analyzed. RESULTS: A total of 1532 stone analyses were performed (992 from males and 540 from females). The mean age was younger in males (p<0.001). Males included more cases with larger BMI, hyperuricemia, and obesity, while females had more urinary tract infections. Multiple components were present in 61.8% of stones. Calcium oxalate (CaOx) (67.0%) was the most common component, followed by uric acid (UA) (11.8%), infection stone (11.4%), calcium phosphate (CaP) (8.0%), cystine (1.1%), brushite (0.4%), and 2,8-dihydroxyadenine (0.2%). Men contributed with more CaOx stones than women at age 30-49 years (all p<0.01) and more UA stones at 30-59 years (all p<0.05). Women contributed with more infection stones than men in age groups 30-49 and 60-69 years (all p<0.05), and more CaP stones at 30-49 years. The prevalence peak was 50-59 years in men and 60-69 years in women. Both genders had the lowest prevalence in adolescence. Prevalence of UA stones increased while that of infection stones decreased with aging in both genders. CONCLUSIONS: Age and sex had a strong association with distribution of stone compositions in this Chinese cohort.

N6-methyladenine DNA Modification in Glioblastoma.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.

A novel mass spectrometry method based on competitive non-covalent interaction for the detection of biomarkers.

We report a novel biosensor platform based on competitive non-covalent interaction between ssDNA and a mass tag towards AuNPs, which detects PSA biomarkers sensitively, observed using MALDI MS. A detection limit of 57 pg mL-1 has been achieved, showing an improvement of two orders of magnitude compared to the traditional spectroscopic method.

Poly(glycine)/graphene oxide modified glassy carbon electrode: Preparation, characterization and simultaneous electrochemical determination of dopamine, uric acid, guanine and adenine.

A novel poly(glycine) (p-GLY)/graphene oxide (GO) composite based sensor (p-GLY/GO) was successfully prepared on glassy carbon electrode by simple electropolymerization. Electrochemical responses of analytes on p-GLY/GO modified electrode were studied by cyclic voltammetry and differential pulse voltammetry. The results demonstrated that p-GLY/GO modified electrode showed a favorable application for the simultaneous determination of dopamine (DA), uric acid (UA), guanine (GU) and adenine (AD). Owing to the synergistic effect of p-GLY and GO, the oxidation peaks of four analytes separated well from each other, and the potential separations of oxidation peaks of DA-UA, UA-GU and GU-AD were large up to 170, 350 and 300 mV, respectively. As-prepared p-GLY/GO modified electrode offered wide linear responses for DA, UA, GU and AD over the ranges of 0.20-62, 0.10-105, 0.15-48 and 0.090-103 µM with detection limits of 0.011, 0.061, 0.026 and 0.030 µM (S/N = 3), respectively. Moreover, p-GLY/GO modified electrode presented favorable selectivity, stability and reproducibility, which was a promising candidate as an electrochemical sensor for the simultaneous determination of DA, UA, GU and AD.

A novel adenine-releasing assay for ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) are toxic enzymes that are mostly biosynthesized by plants. RIPs are N-glycosidases that cleave an essential adenine molecule from the 28S rRNA. This is followed by the irreversible inhibition of protein synthesis leading to cell death. By fusing RIPs to cancer cell specific targeting ligands RIPs have been utilized for targeted anti-tumor therapy. The anti-tumoral efficiency of such conjugates depends significantly on the N-glycosidase activity of the RIP domain. Different methods have been developed in order to determine the N-glycosidase activity of RIPs and RIP domain containing anti-tumor toxins. However the existing methods are elaborate and include radioassays, HPLC and enzymatic conversion assays. Here, a simple and cost effective N-glycosidase assay is presented, which is based on the direct determination of the released adenine by thin-layer chromatography (TLC) and TLC-densitometry. An adenine based single stranded oligonucleotide is used as substrate. Following TLC development the released adenine is quantified on silica glass plates by UV absorbance at 260nm.

Label-free electrochemical DNA biosensor for guanine and adenine by ds-DNA/poly(L-cysteine)/Fe3O4 nanoparticles-graphene oxide nanocomposite modified electrode.

In this study, we aim to design a simple and effective electrochemical DNA biosensor based on a carbon paste electrode modified with ds-DNA/poly(L-cysteine)/Fe3O4 nanoparticles-graphene oxide (ds-DNA/p(L-Cys)/Fe3O4 NPs-GO/CPE) for sensitive detection of adenine (A) and guanine (G). The electrocatalytic oxidation of A and G on the electrode was explored by differential pulse voltammetry (DPV) and cyclic voltammetry (CV). This sensor shows separated and well-defined peaks for A and G, by which one can determine these biological bases individually or simultaneously. The ds-DNA/p(L-Cys)/Fe3O4 NPs-GO/CPE exhibited an increase in peak currents and the electron transfer kinetics and decrease in the overpotential for the oxidation reaction of A and G. Under the optimal conditions a linear relationship is figured out between the peak current and the analytes' concentrations on a range of 0.01-30.0µM and 0.01-25.0µM for simultaneous determination of A and G, with detection limits of 3.48 and 1.59nM, respectively. As well as, individually determination is resulted two linear concentration ranges of 0.01-30.0µM for A and 0.01-25.0µM for G with detection limits of 3.90 and 1.58nM for A and G, respectively. The proposed biosensor exhibited some advantages in terms of simplicity, rapidity, high sensitivity, good reproducibility and long-term stability. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G + C)/(A + T) of DNA was calculated as about 0.77 for various DNA samples. This study also ascertained that the proposed biosensor can be profitable to evaluate DNA bases damage.

High resolution mass approach to characterize refrigerated black truffles stored under different storage atmospheres.

Freshly harvested Tuber melanosporum samples were packed and stored at 4°C under reduced atmospheric pressure or modified atmosphere for four weeks. Multivariate analysis was employed to correlate the antioxidant power of the ethanolic extracts of the samples with the chemical composition determined by high resolution mass spectrometry. High performance liquid chromatography coupled with a coularray detector was applied to select the chemical species associated with the antioxidant power. Four classes of chemical compounds were investigated in more detail by a targeted approach: derivatives of glutathione, adenine (such as S-adenosyl-homocysteine), oxidized linoleic acid and ergosterol. Adducts containing glutathione and adenine with oxidized linoleic acid were observed in TM for the first time and can be considered markers of freshness of the product. S-adenosyl-homocysteine, the acetyl-carnitine adduct with cysteinyl-glycine and several oxidized linoleic acid derivatives were among the markers of degradation.

Poly-l-cysteine/electrospun copper oxide nanofibers-zinc oxide nanoparticles nanocomposite as sensing element of an electrochemical sensor for simultaneous determination of adenine and guanine in biological samples and evaluation of damage to dsDNA and DNA purine bases by UV radiation.

A new nanocomposite film constructed of poly-l-cysteine/zinc oxide nanoparticles-electrospun copper oxide nanofibers (PLC/ZnO-NPs-CuO-NFs) was prepared on the surface of the graphite electrode (GE). The novel electrode was successfully applied for the simultaneous determination of guanine (G) and adenine (A), two of the most important components of DNA and RNA. The PLC/ZnO-NPs-CuO-NFs/GE enhanced the anodic peak currents of the purine bases conspicuously and could determine them sensitively and separately in 0.1 M phosphate buffer solution at the physiological pH (7.0). The synthesized nanofibers, nanoparticles and nanocomposite were characterized by different methods such as Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and energy dispersive X-ray analysis (EDS). Under the optimum operating conditions, linear calibration curves were obtained in the range of 0.05-6.78 and 0.01-3.87 µM with a detection limit of 12.48 and 1.25 nM for G and A, respectively. The proposed method was applied to quantify A and G in three different DNA samples with satisfactory results. In addition, damage to human blood double-stranded DNA (dsDNA) and DNA purine bases (liberated in previously hydrolyzed human blood dsDNA) caused by UV-C and UV-B were evaluated. The results demonstrated that the proposed biosensing platform not only provides a novel and sensitive approach to detecting DNA damage, but also can be used for simultaneous determination of purine bases and major products of DNA oxidative damage.

Structure-Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase.

Base excision repair glycosylases locate and remove damaged bases in DNA with remarkable specificity. The MutY glycosylases, unusual for their excision of undamaged adenines mispaired to the oxidized base 8-oxoguanine (OG), must recognize both bases of the mispair in order to prevent promutagenic activity. Moreover, MutY must effectively find OG:A mismatches within the context of highly abundant and structurally similar T:A base pairs. Very little is known about the factors that initiate MutY's interaction with the substrate when it first encounters an intrahelical OG:A mispair, or about the order of recognition checkpoints. Here, we used structure-activity relationships (SAR) to investigate the features that influence the in vitro measured parameters of mismatch affinity and adenine base excision efficiency by E. coli MutY. We also evaluated the impacts of the same substrate alterations on MutY-mediated repair in a cellular context. Our results show that MutY relies strongly on the presence of the OG base and recognizes multiple structural features at different stages of recognition and catalysis to ensure that only inappropriately mispaired adenines are excised. Notably, some OG modifications resulted in more dramatic reductions in cellular repair than in the in vitro kinetic parameters, indicating their importance for initial recognition events needed to locate the mismatch within DNA. Indeed, the initial encounter of MutY with its target base pair may rely on specific interactions with the 2-amino group of OG in the major groove, a feature that distinguishes OG:A from T:A base pairs. These results furthermore suggest that inefficient substrate location in human MutY homologue variants may prove predictive for the early onset colorectal cancer phenotype known as MUTYH-Associated Polyposis, or MAP.

Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N6-Methyladenine in Human Cells.

DNA N6-methyl-2'-deoxyadenosine (6mdA) is an epigenetic modification in both eukaryotes and bacteria. Here we exploited stable isotope-labeled deoxynucleoside [15N5]-2'-deoxyadenosine ([15N5]-dA) as an initiation tracer and for the first time developed a metabolically differential tracing code for monitoring DNA 6mdA in human cells. We demonstrate that the initiation tracer [15N5]-dA undergoes a specific and efficient adenine deamination reaction leading to the loss the exocyclic amine 15N, and further utilizes the purine salvage pathway to generate mainly both [15N4]-dA and [15N4]-2'-deoxyguanosine ([15N4]-dG) in mammalian genomes. However, [15N5]-dA is largely retained in the genomes of mycoplasmas, which are often found in cultured cells and experimental animals. Consequently, the methylation of dA generates 6mdA with a consistent coding pattern, with a predominance of [15N4]-6mdA. Therefore, mammalian DNA 6mdA can be potentially discriminated from that generated by infecting mycoplasmas. Collectively, we show a promising approach for identification of authentic DNA 6mdA in human cells and determine if the human cells are contaminated with mycoplasmas.

Identification of a Pseudomonas aeruginosa PAO1 DNA Methyltransferase, Its Targets, and Physiological Roles.

DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N6-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in the PAO1 methylation status were dependent on growth conditions and affected P. aeruginosa pathogenicity in a Galleria mellonella infection model. Furthermore, we found that methylated motifs in promoter regions led to shifts in sense and antisense gene expression, emphasizing the role of enzymatic DNA methylation as an epigenetic control of phenotypic traits in P. aeruginosa Since the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the global P. aeruginosa transcriptome and thus may facilitate adaptation to new and challenging habitats.IMPORTANCE With the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in the Galleria mellonella infection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNA prrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of the prrF1 small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria.

The decreased N6-methyladenine DNA modification in cancer cells.

N6-methyladenine (6 mA) is a recently characterized DNA modification in mammalian genomes, although its biological importance remains to be resolved. Using a highly sensitive HPLC/MS/MS approach, here we report regulation of 6 mA modification in mammalian cells. To these aspects, down-regulation of 6 mA modification was first characterized in human cancer cells and tissues, relative to their normal controls. In contrast to the relative stable 5 mC modification, a dramatic decrease of 6 mA modification was found, showing that 6 mA is the most regulated DNA modification in cancers. In addition to the regulation in cancer cells, a hundreds-fold increase of 6 mA modification was found for in vitro cultured human cells, relative to the in vivo cells. This up-regulation was also confirmed with in vitro cultured mouse cells. Taken together, our study revealed distinct 6 mA modification profiles in the cancer and cultured cells. Considering its distinct regulation from that of 5 mC, our study suggests that 6 mA DNA modification may play a crucial role in cell fate transition of mammalian cells.

Continuous Fluorescence Assays for Reactions Involving Adenine.

5'-Methylthioadenosine phosphorylase (MTAP) and 5'-methylthioadenosine nucleosidase (MTAN) catalyze the phosphorolysis and hydrolysis of 5'-methylthioadenosine (MTA), respectively. Both enzymes have low KM values for their substrates. Kinetic assays for these enzymes are challenging, as the ultraviolet absorbance spectra for reactant MTA and product adenine are similar. We report a new assay using 2-amino-5'-methylthioadenosine (2AMTA) as an alternative substrate for MTAP and MTAN enzymes. Hydrolysis or phosphorolysis of 2AMTA forms 2,6-diaminopurine, a fluorescent and easily quantitated product. We kinetically characterize 2AMTA with human MTAP, bacterial MTANs and use 2,6-diaminopurine as a fluorescent substrate for yeast adenine phosphoribosyltransferase. 2AMTA was used as the substrate to kinetically characterize the dissociation constants for three-transition-state analogue inhibitors of MTAP and MTAN. Kinetic values obtained from continuous fluorescent assays with MTA were in good agreement with previously measured literature values, but gave smaller experimental errors. Chemical synthesis from ribose and 2,6-dichloropurine provided crystalline 2AMTA as the oxalate salt. Chemo-enzymatic synthesis from ribose and 2,6-diaminopurine produced 2-amino-S-adenosylmethionine for hydrolytic conversion to 2AMTA. Interaction of 2AMTA with human MTAP was also characterized by pre-steady-state kinetics and by analysis of the crystal structure in a complex with sulfate as a catalytically inert analogue of phosphate. This assay is suitable for inhibitor screening by detection of fluorescent product, for quantitative analysis of hits by rapid and accurate measurement of inhibition constants in continuous assays, and pre-steady-state kinetic analysis of the target enzymes.