Widespread Existence of Cytosine Methylation in Yeast DNA Measured by Gas Chromatography/Mass Spectrometry

DNA methylation is one of the major epigenetic modifications and has been involved in a number of biological processes in mammalian cells. Yeast is widely used as a model organism for studying cell metabolism, cell cycle regulation, and signal transduction. However, it remains controversial whether methylated cytosine (5-methylcytosine, 5mC) exists in the yeast genome. In the current study, we developed a highly sensitive method based on gas chromatography/mass spectrometry (GC/MS) and systematically examined the incidence of 5mC in 19 yeast strains, which represent 16 yeast species. Our results showed that DNA methylation is widespread in yeast and the genome-wide DNA methylation of the studied yeast strains ranged from 0.014 to 0.364%, which were 1 to 2 orders of magnitude lower than that in mammalian cells (i.e., 3–8%). Furthermore, we found that the 5mC content in yeast varied considerably at different growth stages and DNA methylation inhibitor 5-azacytidine could induce a decrease in genome-wide DNA methylation as that in mammalian cells. The demonstration of the universal presence of DNA cytosine methylation in yeast constituted the first and essential step toward understanding the functions of this methylation in yeast

Determination of Phytochelatins in Rice by Stable Isotope Labeling Coupled with Liquid Chromatography–Mass Spectrometry

A highly sensitive method was developed for the detection of phytochelatins (PCs) in rice by stable isotope labeling coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (IL–LC–ESI–MS/MS) analysis. A pair of isotope-labeling reagents [ω-bromoacetonylquinolinium bromide (BQB) and BQB-<i>d</i>⁷] were used to label PCs in plant sample and standard PCs, respectively, and then combined prior to LC/MS analysis. The heavy labeled standards were used as the internal standards for quantitation to minimize the matrix and ion suppression effects in MS analysis. In addition, the ionization efficiency of PCs was greatly enhanced through the introduction of a permanent charged moiety of quaternary ammonium of BQB into PCs. The detection sensitivities of PCs upon BQB labeling improved by 14–750-fold, and therefore, PCs can be quantitated using only 5 mg of plant tissue. Furthermore, under cadmium (Cd) stress, we found that the contents of PCs in rice dramatically increased with the increased concentrations and treatment time of Cd. It was worth noting that PC₅ was first identified and quantitated in rice tissues under Cd stress in the current study. Taken together, this IL–LC–ESI–MS/MS method demonstrated to be a promising strategy in detection of PCs in plants with high sensitivity and reliability

Sensitive and Simultaneous Determination of 5‑Methylcytosine and Its Oxidation Products in Genomic DNA by Chemical Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry Analysis

Cytosine methylation (5-methylcytosine, 5-mC) in genomic DNA is an important epigenetic mark that has regulatory roles in diverse biological processes. 5-mC can be oxidized stepwise by the ten–eleven translocation (TET) proteins to form 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC), which constitutes the active DNA demethylation pathway in mammals. Owing to the extremely limited contents of endogenous 5-mC oxidation products, no reported method can directly determine all these cytosine modifications simultaneously. In the current study, we developed selective derivatization of cytosine moieties with 2-bromo-1-(4-dimethylamino-phenyl)-ethanone (BDAPE) coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the simultaneous determination of these cytosine modifications in genomic DNA. The chemical derivatization notably improved the liquid chromatography separation and dramatically increased detection sensitivities of these cytosine modifications. The limits of detection (LODs) of the derivatives of 5-mC, 5-hmC, 5-foC, and 5-caC were 0.10, 0.06, 0.11, and 0.23 fmol, respectively. Using this method, we successfully quantified 5-mC, 5-hmC, 5-foC, and 5-caC in genomic DNA from human colorectal carcinoma (CRC) tissues and tumor-adjacent normal tissues. The results demonstrated significant depletion of 5-hmC, 5-foC, and 5-caC in tumor tissues compared to tumor-adjacent normal tissues, and the depletion of 5-hmC, 5-foC, and 5-caC may be a general feature of CRC; these cytosine modifications could serve as potential biomarkers for the early detection and prognosis of CRC. Moreover, the marked depletion of 5-hmC, 5-foC, and 5-caC may also have profound effects on epigenetic regulation in the development and formation of CRC

Profiling of Thiol-Containing Compounds by Stable Isotope Labeling Double Precursor Ion Scan Mass Spectrometry

Here we developed a novel strategy of isotope labeling in combination with high-performance liquid chromatography–double precursor ion scan mass spectrometry (IL–LC–DPIS-MS) analysis for nontargeted profiling of thiol-containing compounds. In this strategy, we synthesized a pair of isotope labeling reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-<i>d</i>₇ bromide, BQB-<i>d</i>₇) that contain a reactive group, an isotopically labeled moiety, and an ionizable group to selectively label thiol-containing compounds. The BQB and BQB-<i>d</i>₇ labeled compounds can generate two characteristic product ions <i>m</i>/<i>z</i> 218 and 225, which contain an isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. The peak pairs with characteristic mass differences can be readily extracted from the two precursor ion scan (PIS) spectra and assigned as potential thiol-containing candidates, which facilitates the identification of analytes. BQB and BQB-<i>d</i>₇ labeled thiol-containing compounds can be clearly distinguished by generating two individual ion chromatograms. Thus, thiol-containing compounds from two samples labeled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, offering good identification and accurate quantification by eliminating the MS response fluctuation and mutual interference from the two labeled samples. Using the IL–LC–DPIS-MS strategy, we profiled the thiol-containing compounds in beer and human urine, and 21 and 103 thiol candidates were discovered in beer and human urine, respectively. In addition, 9 and 17 thiol candidates in beer and human urine were successfully identified by further comparison with thiol standards or tandem mass spectrometry analysis. Taken together, the IL–LC–DPIS-MS method is demonstrated to be a promising strategy in the profiling of compounds with identical groups in metabolomics study

Facile Preparation of SiO₂/TiO₂ Composite Monolithic Capillary Column and Its Application in Enrichment of Phosphopeptides

A novel SiO₂/TiO₂ composite monolithic capillary column was prepared by sol–gel technology and successfully applied to enrich phosphopeptides as a metal oxide affinity chromatography (MOAC) material. For the monolith preparation, tetramethoxysilane (TMOS) and tetrabutoxytitanium (TBOT) were used as silica and titania source, respectively, and glycerol was introduced to attenuate the activity of titanium precursor, which provided a mild synthetic condition. The prepared monolith was characterized by energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results revealed an approximate 1/2 molar ratio of titanium to silica as well as an atom-scale homogeneity in the framework. The scanning electron microscopy (SEM) results demonstrated an excellent anchorage between the column and the inner capillary wall, and nitrogen adsorption–desorption experiments showed a bimodal porosity with a narrow mesopore distribution around 3.6 nm. The prepared monolith was then applied for selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA) as well as human blood serum, nonfat milk, and egg white using an in-tube solid phase microextraction (SPME) system. Our results showed that SiO₂/TiO₂ composite monolithic capillary column could efficiently enrich the phosphopeptides from complex matrixes. To the best of our knowledge, this is the first attempt for preparing the silica–metal composite monolithic capillary column, which offers the promising application of the monolith on phosphoproteomics study

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

Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

<div><p>Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography – mass spectrometry (<i>c</i>LC-MS) method for the sensitive assay of <i>in-vitro</i> recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA₁, GA₄, GA₉, GA₂₀). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-<i>co</i>-divinylbenzene-<i>co</i>-ethylene glycol dimethacrylate)(META-<i>co</i>-DVB-<i>co</i>-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62–0.90 fmol. We determined the kinetic parameters (<i>K</i>_m) of recombinant GA3-oxidase in <i>Escherichia coli</i> (<i>E. coli</i>) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA₉ to GA₄ in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology.</p></div

Determination of Oxidation Products of 5‑Methylcytosine in Plants by Chemical Derivatization Coupled with Liquid Chromatography/Tandem Mass Spectrometry Analysis

Cytosine methylation (5-methylcytosine, 5-mC) in DNA is an important epigenetic mark that has regulatory roles in various biological processes. In plants, active DNA demethylation can be achieved through direct cleavage by DNA glycosylases, followed by replacement of 5-mC with cytosine by base excision repair (BER) machinery. Recent studies in mammals have demonstrated 5-mC can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC) by Ten–eleven translocation (TET) proteins. The consecutive oxidations of 5-mC constitute the active DNA demethylation pathway in mammals, which raised the possible presence of oxidation products of 5-mC (5-hmC, 5-foC, and 5-caC) in plant genomes. However, there is no definitive evidence supporting the presence of these modified bases in plant genomic DNA, especially for 5-foC and 5-caC. Here we developed a chemical derivatization strategy combined with liquid chromatography–electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method to determine 5-formyl-2′-deoxycytidine (5-fodC) and 5-carboxyl-2′-deoxycytidine (5-cadC). Derivatization of 5-fodC and 5-cadC by Girard’s reagents (GirD, GirT, and GirP) significantly increased the detection sensitivities of 5-fodC and 5-cadC by 52–260-fold. Using this method, we demonstrated the widespread existence of 5-fodC and 5-cadC in genomic DNA of various plant tissues, indicating that active DNA demethylation in plants may go through an alternative pathway similar to mammals besides the pathway of direct DNA glycosylases cleavage combined with BER. Moreover, we found that environmental stresses of drought and salinity can change the contents of 5-fodC and 5-cadC in plant genomes, suggesting the functional roles of 5-fodC and 5-cadC in response to environmental stresses

Characterizations of the META-silica hybrid monolithic column.

<p>(A) – (C) SEM images. (A) ×1,000 wide-view, (B) ×4,000 close-up-view, and (C) ×15,000 close-up-view. (D) The N₂ isothermal plot with the inset showing the pore-size distribution. (E) The effect of flow rate on the back pressure of the monolithic column. (F) Van Deemter plot of the height equivalent to a theoretical plate as a function of flow rate. Experimental conditions: column, poly(META-<i>co</i>-DVB-<i>co</i>-EDMA) monolithic column (30-cm long, 100 µm <i>i.d.</i>, 360 µm <i>o.d.</i>); UV detection wavelength, 254 nm for acrylamide, 214 nm for thiourea and benzene; mobile phase used in (E), ACN; mobile phase used in (F), ACN/H₂O (60/40, v/v).</p

Mutagenic and Cytotoxic Properties of Oxidation Products of 5-Methylcytosine Revealed by Next-Generation Sequencing

<div><p>5-methylcytosine (5-mC) can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and finally to 5-carboxylcytosine (5-caC), which is thought to function in active DNA cytosine demethylation in mammals. Although the roles of 5-mC in epigenetic regulation of gene expression are well established, the effects of 5-hmC, 5-foC and 5-caC on DNA replication remain unclear. Here we report a systematic study on how these cytosine derivatives (5-hmC, 5-foC and 5-caC) perturb the efficiency and accuracy of DNA replication using shuttle vector technology in conjugation with next-g sequencing. Our results demonstrated that, in <i>Escherichia coli</i> cells, all the cytosine derivatives could induce CT transition mutation at frequencies of 0.17%–1.12%, though no effect on replication efficiency was observed. These findings provide an important new insight on the potential mutagenic properties of cytosine derivatives occurring as the intermediates of DNA demethylation.</p></div