Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms

Wang, Xiaoli; Suo, Yongshan; Yin, Ruichuan; Shen, Heqing; Wang, Hailin

doi:10.1016/j.jchromb.2011.04.002

Cited by 34 publications

(36 citation statements)

References 38 publications

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“…UPLC-MS/MS analysis was performed according to a previously published method [43]. Genomic DNA (0.2 μg) extracted from Ad and NBL was digested with 1U DNase I, 2U Alkaline Phoaphatase, Calf Intestinal (CIP) and 0.005U snake venom phosphodiesterase I at 37°C for 24 h. A microcon centrifugal filter device with a 3,000 D cutoff membrane was used to remove protein from the digested DNA samples by centrifuging at 12,000 rpm for 60 minutes.…”

Section: Methodsmentioning

confidence: 99%

Differential DNA methylation in discrete developmental stages of the parasitic nematode Trichinella spiralis

Gao

Liu

et al. 2012

Genome Biol

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BackgroundDNA methylation plays an essential role in regulating gene expression under a variety of conditions and it has therefore been hypothesized to underlie the transitions between life cycle stages in parasitic nematodes. So far, however, 5'-cytosine methylation has not been detected during any developmental stage of the nematode Caenorhabditis elegans. Given the new availability of high-resolution methylation detection methods, an investigation of life cycle methylation in a parasitic nematode can now be carried out.ResultsHere, using MethylC-seq, we present the first study to confirm the existence of DNA methylation in the parasitic nematode Trichinella spiralis, and we characterize the methylomes of the three life-cycle stages of this food-borne infectious human pathogen. We observe a drastic increase in DNA methylation during the transition from the new born to mature stage, and we further identify parasitism-related genes that show changes in DNA methylation status between life cycle stages.ConclusionsOur data contribute to the understanding of the developmental changes that occur in an important human parasite, and raises the possibility that targeting DNA methylation processes may be a useful strategy in developing therapeutics to impede infection. In addition, our conclusion that DNA methylation is a mechanism for life cycle transition in T. spiralis prompts the question of whether this may also be the case in any other metazoans. Finally, our work constitutes the first report, to our knowledge, of DNA methylation in a nematode, prompting a re-evaluation of phyla in which this epigenetic mark was thought to be absent.

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Section: Methodsmentioning

confidence: 99%

Differential DNA methylation in discrete developmental stages of the parasitic nematode Trichinella spiralis

Gao

Liu

et al. 2012

Genome Biol

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“…The assay provided accurate levels, could be performed in 96‐well plate formats (to therefore allow high‐throughput analysis), and demonstrated a similar sensitivity in full‐scan high‐resolution mass analysis on an Orbitrap MS compared to MRM sensitivity levels. Other assays used UPLC type separations in order to improve throughput for DNA methylation analysis (Wang et al, ). UPLC‐MRM analysis was optimized for the study of DNA methylation in human sperm as an indicator of infertility, and used a DNA methylation inhibitor to validate methylation level changes during development.…”

Section: Dna Methylation Analysismentioning

confidence: 99%

Mass spectrometry analysis of nucleosides and nucleotides

Dudley

Bond

2013

Mass Spec Rev

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Mass spectrometry has been widely utilised in the study of nucleobases, nucleosides and nucleotides as components of nucleic acids and as bioactive metabolites in their own right. In this review, the application of mass spectrometry to such analysis is overviewed in relation to various aspects regarding the analytical mass spectrometric and chromatographic techniques applied and also the various applications of such analysis.

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“…To our knowledge, the global methylation analysis of sperm DNA has only been described in eight studies 1718192021222324. The methodology used for the evaluation of global sperm DNA methylation has been limited to a few techniques, most often based on immunofluorescent or colorimetric measurements, followed by microscopic validation or flow cytometry 171819202124.…”

Section: Introductionmentioning

confidence: 99%

“…The methodology used for the evaluation of global sperm DNA methylation has been limited to a few techniques, most often based on immunofluorescent or colorimetric measurements, followed by microscopic validation or flow cytometry 171819202124. Only two studies have described methods based on chromatographic measurement, i.e., ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) or high-performance liquid chromatography (HPLC) 2223…”

Section: Introductionmentioning

confidence: 99%

Global methylation status of sperm DNA in carriers of chromosome structural aberrations

et al. 2017

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Male infertility might be clearly associated with aberrant DNA methylation patterns in human spermatozoa. An association between oxidative stress and the global methylation status of the sperm genome has also been suggested. The aim of the present study was to determine whether the global sperm DNA methylation status was affected in the spermatozoa of carriers of chromosome structural aberrations. The relationships between the 5-methylcytosine (m5C) levels in spermatozoa and chromatin integrity status were evaluated. The study patients comprised male carriers of chromosome structural aberrations with reproductive failure (n = 24), and the controls comprised normozoospermic sperm volunteers (n = 23). The global m5C level was measured using thin-layer chromatography (TLC) and immunofluorescence (IF) techniques. The sperm chromatin integrity was assessed using aniline blue (AB) staining and TUNEL assay. The mean m5C levels were similar between the investigated chromosome structural aberrations carriers (P) and controls (K). However, sperm chromatin integrity tests revealed significantly higher values in chromosomal rearrangement carriers than in controls (P < 0.05). Although the potential relationship between sperm chromatin integrity status and sperm DNA fragmentation and the m5C level juxtaposed in both analyzed groups (P vs K) was represented in a clearly opposite manner, the low chromatin integrity might be associated with the high hypomethylation status of the sperm DNA observed in carriers of chromosome structural aberrations.

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Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms

Cited by 34 publications

References 38 publications

Differential DNA methylation in discrete developmental stages of the parasitic nematode Trichinella spiralis

Differential DNA methylation in discrete developmental stages of the parasitic nematode Trichinella spiralis

Mass spectrometry analysis of nucleosides and nucleotides

Global methylation status of sperm DNA in carriers of chromosome structural aberrations

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