Profiling of epigenetic DNA modifications by advanced liquid chromatography-mass spectrometry technologies

Lai, Weiyi; Mo, Jiezhen; Yin, Jie; Lyu, Cong; Wang, Hailin

doi:10.1016/j.trac.2018.10.031

Cited by 30 publications

(34 citation statements)

References 108 publications

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“…Mass spectrometry (MS) has been widely used to detect and quantify nucleic acid modifications as MS exhibits high detection sensitivity and good capability to identify compounds. 52–58 LC/MS is the preferred method for the sensitive detection and quantification of the global levels of modifications in genomic DNA. Typically, DNA is enzymatically digested into 2′-deoxynucleosides or 2′-deoxynucleotides and then determined by LC/MS.…”

Section: Methods For Quantification Of Dna Modificationsmentioning

confidence: 99%

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Quantification and mapping of DNA modifications

Dai

Yuan

2021

RSC Chem. Biol.

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Section: Methods For Quantification Of Dna Modificationsmentioning

confidence: 99%

Section: Liquid Chromatography/mass Spectrometrymentioning

confidence: 99%

Quantification and mapping of DNA modifications

Dai

Yuan

2021

RSC Chem. Biol.

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“…An Agilent 1290 II UHPLC system coupled with ESI-triple quadrupole mass spectrometers 6470 and 6410 (Agilent Technologies, U.S.A.) was respectively applied to the detection of 6mA (m/z 266–150) and DNA Cytosine (dC) (m/z 228–112). The conditions for chromatography and mass spectrometry were as we reported previously [ 13 , 14 , 16 , 23 ].…”

Section: Methodsmentioning

confidence: 99%

“…However, largely due to the low-level presence of 6mA, the distribution and functions of 6mA in eukaryotic genomic DNA are still elusive [ 2 ]. As one of the most sensitive detection methods of DNA modifications, ultra-high-performance liquid chromatography-quadruple mass spectrometry (UHPLC-MS/MS) has been widely applied to the study of 6mA [ 13 , 14 ]. In vitro activity analysis of proteins coupled with UHPLC-MS/MS analysis is a common research strategy.…”

Section: Introductionmentioning

confidence: 99%

Conjoint expression and purification strategy for acquiring proteins with ultra-low DNA N6-methyladenine backgrounds in Escherichia coli

2021

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DNA N6-methyladenine (6mA), a kind of DNA epigenetic modification, is widespread in eukaryotes and prokaryotes. An enzyme activity study coupled with 6mA detection using ultra-high-performance liquid chromatography-quadruple mass spectrometry (UHPLC-MS/MS) is commonly applied to investigate 6mA potentially related enzymes in vitro. However, the protein expressed in a common Escherichia coli (E. coli) strain shows an extremely high 6mA background due to a minute of co-purified bacterial DNA, though it has been purified to remove DNA using multiple strategies. Furthermore, as occupied by DNA with abundant 6mA, the activity of 6mA related proteins will be influenced seriously. Here, to address this issue, we for the first time construct a derivative of E. coli Rosetta (DE3) via the λRed knockout system specifically for the expression of 6mA related enzymes. The gene dam encoding the 6mA methyltransferase (MTase) is knocked out in the newly constructed strain named LAMBS (low adenine methylation background strain). Contrasting with E. coli Rosetta (DE3), LAMBS shows an ultra-low 6mA background on the genomic DNA when analyzed by UHPLC-MS/MS. We also demonstrate an integral strategy of protein purification, coupled with the application of LAMBS. As a result, the purified protein expressed in LAMBS exhibits an ultra-low 6mA background comparing with the one expressed in E. coli Rosetta (DE3). Our integral strategy of protein expression and purification will benefit the in vitro investigation and application of 6mA related proteins from eukaryotes, although these proteins are elusive until now.

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“…Because of the high detection sensitivity and good capability for deciphering structures, LC-MS recently has become a more widely used platform for analyzing lowabundant nucleic acid modications. 26,33,34 In the current study, we established a liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/ MS) method to determine the dimethylation of cytidines, m 3 Cm, m 4 Cm and m 5 Cm. Under optimized analytical conditions, m 3 Cm, m 4 Cm and m 5 Cm can be clearly distinguished.…”

Section: Introductionmentioning

confidence: 99%