Sensitive analysis of trehalose-6-phosphate and related sugar phosphates in plant tissues by chemical derivatization combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Trehalose-6-phosphate (T6P) is an important signaling metabolite that is involved in many physiological processes. However, the mechanism of the biological functions of T6P is not fully understood. Quantification of T6P in plants will be beneficial to elucidate the mechanism. However, it is still a challenge to chromatographically separate and sensitively detect T6P and related sugar phosphates. In the current study, we developed a method for effective separation and sensitive detection of glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), sucrose-6-phosphate (S6P) and T6P in plant tissues by chemical derivatization combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry (ChD-HILIC-MS/MS). With this method, two pairs of isomers (G1P/G6P and S6P/T6P) could be well separated on a HILIC column and sensitively detected by MS with limits of detection (LODs) ranging from 0.1 to 0.6 ng mL-1. The developed method was successfully applied to the detection of endogenous G1P, G6P, S6P and T6P in small amounts of plant tissues, such as 1 mg fresh weight of Oryza sativa shoot.

Modified nucleoside triphosphates exist in mammals.

DNA and RNA contain diverse chemical modifications that exert important influences in a variety of cellular processes. In addition to enzyme-mediated modifications of DNA and RNA, previous in vitro studies showed that pre-modified nucleoside triphosphates (NTPs) can be incorporated into DNA and RNA during replication and transcription. Herein, we established a chemical labeling method in combination with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis for the determination of endogenous NTPs in the mammalian cells and tissues. We synthesized 8-(diazomethyl)quinoline (8-DMQ) that could efficiently react with the phosphate group under mild condition to label NTPs. The developed method allowed sensitive detection of NTPs, with the detection limits improved by 56-137 folds. The results showed that 12 types of endogenous modified NTPs were distinctly determined in the mammalian cells and tissues. In addition, the majority of these modified NTPs exhibited significantly decreased contents in human hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues. Taken together, our study revealed the widespread existence of various modified NTPs in eukaryotes.

Determination of formylated DNA and RNA by chemical labeling combined with mass spectrometry analysis.

Nucleic acids carry diverse chemical modifications that exert critical influences in a variety of cellular processes in living organisms. In addition to methylation, the emerging DNA and RNA formylation has been reported to play functional roles in various physiological processes. However, the amounts of formylated DNA and RNA are extremely low and detection of DNA and RNA formylation is therefore a challenging task. To address this issue, we developed a strategy by chemical labeling combined with in-tube solid-phase microextraction - ultra high performance liquid chromatography - electrospray ionization - tandem mass spectrometry (in-tube SPME-UPLC-ESI-MS/MS) analysis for the sensitive determination of DNA and RNA formylation. Using the developed method, we were able to simultaneously measure six formylated nucleosides, including 5-formyl-2'-deoxycytidine (5-fodC), 5-formylcytidine (5-forC), 5-formyl-2'-deoxyuridine (5-fodU), 5-formyluridine (5-forU), 2'-O-methyl-5-formylcytidine (5-forCm) and 2'-O-methyl-5- formyluridine (5-forUm), from DNA and RNA of cultured human cells and multiple mammalian tissues. The detection limits of these formylated nucleosides improved by 307-884 folds using Girard's P (GirP) labeling coupled with in-tube SPME-UPLC-ESI-MS/MS analysis. It was worth noting that 5-forU, 5-forCm and 5-forUm which have not been detected in human sample before, were discovered in cultured human cells and tissues in the current study. In addition, we observed significant increase of 5-forC and 5-forU in RNA (p = 0.027 for 5-forC; p = 0.028 for 5-forU) and 5-fodU in DNA (p = 0.002) in human thyroid carcinoma tissues compared to normal tissues adjacent to the tumor using synthesized stable isotope GirP (d5-GirP)-assisted quantification. Our results indicated that aberrant DNA and RNA formylation may contribute to the tumor formation and development. In addition, monitoring of DNA and RNA formylation may also serve as indicator for cancer diagnostics. Taken together, the developed chemical labeling combined with in-tube SPME-UPLC-ESI-MS/MS analysis can facilitate the in-depth functional study of DNA and RNA formylation.

Formation and Determination of Endogenous Methylated Nucleotides in Mammals by Chemical Labeling Coupled with Mass Spectrometry Analysis.

5-Methylcytosine (5-mC) is an important epigenetic mark that plays critical roles in a variety of cellular processes. To properly exert physiological functions, the distribution of 5-mC needs to be tightly controlled in both DNA and RNA. In addition to methyltransferase-mediated DNA and RNA methylation, premethylated nucleotides can be potentially incorporated into DNA and RNA during replication and transcription. To exclude the premodified nucleotides into DNA and RNA, endogenous 5-methyl-2'-deoxycytidine monophosphate (5-Me-dCMP) generated from nucleic acids metabolism can be enzymatically deaminated to thymidine monophosphate (TMP). Therefore, previous studies failed to detect 5-Me-dCMP or 5-methylcytidine monophosphate (5-Me-CMP) in cells. In the current study, we established a method by chemical labeling coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5-Me-dCMP and 5-Me-CMP. As N,N-dimethyl-p-phenylenediamine (DMPA) was utilized for labeling, the detection sensitivities of nucleotides increased by 88-372-fold due to the introduction of a tertiary amino group and a hydrophobic moiety from DMPA. Using this method, we found that endogenous 5-Me-dCMP and 5-Me-CMP widely existed in cultured human cells, human tissues, and human urinary samples. The presence of endogenous 5-Me-dCMP and 5-Me-CMP indicates that deaminases may not fully deaminate these methylated nucleotides. Consequently, the remaining premethylated nucleosides could be converted to nucleoside triphosphates as building blocks for DNA and RNA synthesis. Furthermore, we found that the contents of 5-Me-dCMP and 5-Me-CMP exhibited significant decreases in renal carcinoma tissues and urine samples of lymphoma patients compared to their controls, probably due to more reutilization of methylated nucleotides in DNA and RNA synthesis. This study is, to the best of our knowledge, the first report for detecting endogenous 5-Me-dCMP and 5-Me-CMP in mammals. The detectable endogenous methylated nucleotides indicate the potential deleterious effects of premodified nucleotides on aberrant gene regulation in cancers.

Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two folds in both gastric cancer and lymphoma; one ribose conjugate increased more than two folds in both esophagus cancer and lymphoma. The developed analytical method provides a good platform to study the modified ribonucleosides in human body fluids.

DNA hydroxymethylation age of human blood determined by capillary hydrophilic-interaction liquid chromatography/mass spectrometry.

BACKGROUND: Aging is a complex phenomenon and characterized by a progressive decline in physiology and function of adult tissues. However, it hasn't been well established of the correlation between aging and global DNA methylation and hydroxymethylation that regulate the growth and development of higher organisms. RESULTS: We developed an on-line trapping/capillary hydrophilic-interaction liquid chromatography/electrospray ionization-mass spectrometry method for ultra-sensitive and simultaneous quantification of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in genomic DNA from human blood. Limits of detection for 5-mC and 5-hmC were 0.04 and 0.13 fmol, respectively. The imprecision and recovery of the method were determined with the relative standard deviations (RSDs) and relative errors being <11.2 and 14.0 %, respectively. We analyzed the contents of 5-mC and 5-hmC in genomic DNA of blood from 238 healthy people aged from 1 to 82 years. The results showed that 5-hmC content was significantly decreased and highly correlated with aging process, while 5-mC only showed slight correlation with age. We then established a DNA hydroxymethylation age model according to 5-hmC content with a mean absolute deviation (MAD) of approximate 8.9 years. We also calculated the mean relative error (MRE) using the predicted ages based on the age model and the chronological ages. The results showed that the MRE was 18.3 % for samples with ages from 20 to 82 years (95 % confidence interval, N = 190). CONCLUSIONS: The global DNA hydroxymethylation represents a strong and reproducible mark of chronological age, which could be potentially applied in health assessment and prevention of diseases. The identification of biological or environmental factors that influence DNA hydroxymethylation aging rate may permit quantitative assessments of their impacts on health.

Metal Oxide-Based Selective Enrichment Combined with Stable Isotope Labeling-Mass Spectrometry Analysis for Profiling of Ribose Conjugates.

Some modified ribonucleosides in biological fluids have been evaluated as cancer-related metabolites. Detection of endogenous modified ribonucleosides in biological fluids may serve as a noninvasive cancers diagnostic method. However, determination of modified ribonucleosides is still challenging because of their low abundance and serious matrix interferences in biological fluids. Here, we developed a novel strategy for comprehensive profiling of ribose conjugates from biological fluids using metal oxide-based dispersive solid-phase extraction (DSPE) followed with in vitro stable isotope labeling and double neutral loss scan-mass spectrometry analysis (DSPE-SIL-LC-DNLS-MS). Cerium dioxide (CeO2) was used to selectively recognize and capture ribose conjugates from complex biological samples under basic environment. The enriched ribose conjugates were subsequently labeled with a pair of isotope labeling reagents (acetone and acetone-d6). The glucosidic bond of acetone labeled ribose conjugates is readily ruptured, and the generated ribose that carries an isotope tag can be lost as a neutral fragment under collision induced dissociation (CID). Since the light (acetone) and heavy (acetone-d6) labeled compounds have the same chemical structures and can generate different neutral loss fragments (NL 172 and 178 Da), it is therefore highly convenient to profile ribose conjugates by double neutral loss scan mode in mass spectrometry analysis. In this respect, the light and heavy labeled compounds were ionized at the same condition but recorded separately on MS spectra, which can significantly improve the detection specificity and facilitate the identification of ribose conjugates. Using the developed DSPE-SIL-LC-DNLS-MS strategy, we profiled the ribose conjugates in human urine, and 49 ribose conjugates were readily identified, among which 7 ribose conjugates exhibited significant contents change between healthy controls and lymphoma patients. The DSPE-SIL-LC-DNLS-MS strategy combines the selective enrichment, stable isotope labeling, and double neutral loss scan - MS analysis, which therefore can efficiently minimize false positive results, facilitate the relative quantification, and notably increase the numbers of identified ribose conjugates in biological fluids samples. Taken together, this study established a promising strategy for the effective profiling of urinary modified ribonucleosides, and simultaneous evaluation of the contents change of multiple modified ribonucleosides should provide more accurate and conclusive results for the use of urinary modified ribonucleosides as indicators of cancers.

Profiling of cis-diol-containing nucleosides and ribosylated metabolites by boronate-affinity organic-silica hybrid monolithic capillary liquid chromatography/mass spectrometry.

RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5'-deoxy-5'-methylthioadensine, N(4)-acetylcytidine, 1-ribosyl-N-propionylhistamine and N(2),N(2),7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers.

Facile one-pot synthesis of a aptamer-based organic-silica hybrid monolithic capillary column by "thiol-ene" click chemistry for detection of enantiomers of chemotherapeutic anthracyclines.

In the current study, we developed a facile strategy for the one-pot synthesis of an aptamer-based organic-silica hybrid monolithic capillary column. A 5'-SH-modified aptamer, specifically targeting doxorubicin, was covalently modified in the hybrid silica monolithic column by a sol-gel method combined with "thiol-ene" click reaction. The prepared monolithic column had good stability and permeability, large specific surface, and showed excellent selectivity towards chemotherapeutic anthracyclines of doxorubicin and epirubicin. In addition, the enantiomers of doxorubicin and epirubicin can be easily separated by aptamer-based affinity monolithic capillary liquid chromatography. Furthermore, doxorubicin and epirubicin spiked in serum and urine were also successfully determined, which suggested that the complex biological matrix had a negligible effect on the detection of doxorubicin and epirubicin. Finally, we quantified the concentration of epirubicin in the serum of breast cancer patients treated with epirubicin by intravenous injection. The developed analytical method is cost-effective and rapid, and biological samples can be directly analyzed without any tedious sample pretreatment, which is extremely useful for monitoring medicines in serum and urine for pharmacokinetic studies.

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.