Determination of methylation specificity of sequence-specific DNA methyltransferases using matrix assisted laser desorption/ionization time-of-flight mass spectrometry | Zendy

Takashi Tamura | Zendy

Open Access

Determination of methylation specificity of sequence-specific DNA methyltransferases using matrix assisted laser desorption/ionization time-of-flight mass spectrometry

Author(s) -

Takashi Tamura

Publication year - 1997

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/25.20.4162

Subject(s) - haeiii , mass spectrometry , oligonucleotide , ecori , biology , sample preparation in mass spectrometry , restriction enzyme , methyltransferase , dna , methylation , protein mass spectrometry , microbiology and biotechnology , chromatography , hpaii , matrix assisted laser desorption/ionization , biochemistry , dna methylation , chemistry , desorption , polymerase chain reaction , tandem mass spectrometry , gene , electrospray ionization , restriction fragment length polymorphism , adsorption , gene expression , organic chemistry

We describe here a sensitive and straightforward method for characterizing the methylation specificity of type II DNA methyltransferase (MTase) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA substrate, prepared by ligation of a commercially available oligonucleotide, was modified by the subject MTase, and was derivatized to a mixture of single-stranded oligonucleotides through endonuclease treatment, heat-denaturation and limited digestion by 3'-terminus-specific phosphodiesterase I. MALDI-TOF mass spectrometry was used to determine the mass differences between the digestion products, and the methylated nucleotide was explicitly identified by the mass increase of 14 Da due to the base modification. The method was applicable to the three representative MTases M. Eco RI, M. Bam HI and M. Hae III.

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