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Abstract Ultraviolet (UV) radiation induces mutagenicity and cytotoxicity in human cells by the formation of DNA lesions, including cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), mainly on thymine-thymine (TpT) dinucleotides. Here, we firstly synthesized the two TpT dimeric lesions with satisfactory yields using a unique UV-irradiated water droplet approach followed by HPLC purification. By the use of purified TpT lesions as standards, we further developed and optimized a quantitative UHPLC-Q-TOF/MS method for the detection of CPDs and 6-4PPs. After the optimization of the enzyme composition and the pH values of hydrolysis solution, a combination of snake venom phosphodiesterase, nuclease P1, and calf intestine alkaline phosphatase can be used for one-step enzymatic digestion to efficiently release the dimeric lesions (CPDs and 6-4PPs) from the genomic DNA. By the use of the one-step digestion and UHPLC-Q-TOF/MS assay for scanning all dimeric lesions, we demonstrate that only are TpT dimeric lesions detectable in genomic DNA of HCT116 cells upon UVC irradiation. The estimated frequency of the CPD of TpT increases from 28.7 to 409 per 106 bases with increasing UVC dosage from 40 J/m2 to 1200 J/m2, while the 6-4PP of TpT increases from 3.7 to 54 per 106 bases. The proposed UHPLC-Q-TOF/MS method is promising for accurate identification and quantitative detection of UV-induced dimeric lesions in cellular DNA. Highlights • Unique UV-irradiated water droplet approach followed by HPLC purification was developed to prepare TpT dimer standards. • A simplified one-step enzymatic process to release TpT dimeric lesions from genomic DNA. • TT CPD and T(6-4)T levels were detected by UHPLC-Q-TOF/MS in HCT116 cells upon UVC-irradiation. [ABSTRACT FROM AUTHOR]

8-Hydroxydeoxyguanosine (8-OHdG) is a widely-used biomarker of oxidative DNA damages. 8-OHdG in peripheral blood leukocyte is associated with mutation and cancer risk. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress rather than that in urine. Accurate identification and quantification of leukocyte 8-OHdG are essential for understanding its mechanism of formation, repair, and biological consequences. In this study, a fast and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated to detect 8-OHdG in human peripheral leukocyte. DNA in blood samples were extracted and digested, then subjected onto UHPLC–MS/MS using an isocratic elution on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm). Multiple reaction monitoring (MRM) mode was adopted by using [ 15 N 5 ]-8-OHdG as an internal standard. The assay was linear over the concentration range of 1.0–100 nM with R 2 = 0.999. The accuracy for spiked samples was 90.9%–94.8%, and the intra-day precision was within 3.7%. The limit of detection (LOD) is 0.30 nM and limit of quantification (LOQ) is 1.0 nM with 5 μl of sample injection. By the analysis of human leukocyte 8-OHdG (n = 121) using the developed UHPLC–MS/MS method, it demonstrates that the level of leukocyte 8-OHdG of the cancer patients group (n = 46) is significantly higher than that of the health control (n = 75). [ABSTRACT FROM AUTHOR]

A sensitive and accurate stable isotope dilution UHPLC–MS/MS method was developed and validated for the detection and quantification of ProdG adducts in human urine, a surrogate for the ProdG adducts in genomic DNA of human. A specific solid phase extraction (SPE) approach was established for selective enrichment of urinary ProdG adducts and elimination of urinary matrix facilitating the coupled MS/MS detection. The recovery of the method is estimated about 84.8–107.2%, and the precision are about 0.8–3.6% for intraday and 2.8–10.0% for interday. Due to that the matrix effect is efficiently eliminated by SPE pretreatment, the limits of detection (LODs, S/N = 3) and quantification (LOQs, S/N = 10) are decreased to 100 and 300 amol for urinary ProdG adducts, respectively. By coupling the developed SPE pretreatment with the UHPLC–MS/MS analysis, ProdG adducts were accurately quantified in healthy human urine. [ABSTRACT FROM AUTHOR]

Abstract: A DNA aptamer based high-performance affinity chromatography is developed for selective extraction and screening of a basic protein lysozyme. First, a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic column was synthesized in situ by thermally initiated radical polymerization, and then an anti-lysozyme DNA aptamer was covalently immobilized on the surface of the monolith through a 16-atom spacer arm. The target protein lysozyme but non-target proteins can be trapped by the immobilized anti-lysozyme DNA aptamer. In contrast, lysozyme cannot be trapped by the immobilized oligodeoxynucleotide that does not contain the sequence of the anti-lysozyme DNA aptamer. The study clearly demonstrates the trapping of lysozyme by the immobilized anti-lysozyme DNA aptamer is mainly due to specific recognition rather than simple electrostatic interaction of positively charged protein and the negatively charged DNA. The inter-day precision was determined as 0.8% for migration time and 4.2% for peak area, respectively. By the use of aptamer affinity monolith, a screening strategy is developed to selectively extract lysozyme from chicken egg white, showing the advantages of high efficiency, low cost and ease-of-operation. [Copyright &y& Elsevier]

Abstract: Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility. We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method for rapid, sensitive, and specific detection of global DNA methylation level in human sperms. Multiple-reaction monitoring (MRM) mode was used in MS/MS detection for accurate quantification of DNA methylation. The intra-day and inter-day precision values of this method were within 1.50–5.70%. By using 2-deoxyguanosine as an internal standard, UPLC–MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines. DNA methyltransferases inhibitor 5-aza-2′-deoxycytidine can significantly reduce global DNA methylation levels in treated cell lines, showing the reliability of our method. We further examined global DNA methylation levels in human sperms, and found that global methylation values varied from 3.79% to 4.65%. The average global DNA methylation level of sperm samples washed only by PBS (4.03%) was relatively lower than that of sperm samples in which abnormal and dead sperm cells were removed by density gradient centrifugation (4.25%), indicating the possible aberrant DNA methylation level in abnormal sperm cells. Clinical application of UPLC–MS/MS method in global DNA methylation detection of human sperms will be useful in human sperm quality evaluation and the study of epigenetic mechanisms responsible for male infertility. [Copyright &y& Elsevier]

Abstract: An ultra-performance liquid chromatography tandem mass spectrometry with multiple reaction monitoring method (UPLC-MRM/MS) is developed for fast and sensitive analysis of four genotoxic stereoisomers of anti-benzo[a]pyrene diol epoxide (BPDE)–N 2dG adducts (trans-(+), trans-(−), cis-(+) and cis-(−)), which result from environmental exposure to ubiquitous pollutant benzo[a]pyrene (B[a]P). The developed method displays a low limit of detection of <0.7fmol (S/N=3) for the four stereoisomers of anti-BPDE–N 2dG, a dynamic range of 2 orders of magnitude (2.3–630fmol, R 2 ≥0.997), and one separation of 2–4min. The developed method enables us to use the stereoisomers of anti-BPDE–N 2dG as a biomarker and to study the stereoselectivity of metabolic activation of B[a]P in human lung A549 cells. The UPLC-MRM/MS analysis of cellular DNA exposed to B[a]P show that activation of B[a]P in A549 cells predominantly induces trans-(+)-anti-BPDE–N 2dG with cis-(+)-anti-BPDE–N 2dG and one syn-BPDE–N 2dG as two minorities, while trans-(−)-anti-BPDE–N 2dG and cis-(−)-anti-BPDE–N 2dG are absent. The observed preferential formation of trans-(+)-anti-BPDE–N 2dG in B[a]P treated A549 cells may result from combined stereoselectivity of the metabolic activation of B[a]P and the reaction of anti-BPDE with dsDNA. The results also suggest that a number of key optical intermediates are formed during activation of B[a]P in A549 cells, including trans-(+)-B[a]P-7,8-dihydrodiol and trans-(−)-B[a]P-7,8-dihydrodiol and their corresponding downstream metabolites (+)-anti-BPDE and (+)-syn-BPDE. [Copyright &y& Elsevier]