In Situ Methylation of Nucleic Acids Using Pyrolysis/Mass Spectrometry

✍ Scribed by Christy Abbas-Hawks; Kent J. Voorhees; Ted L. Hadfield

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 487 KB
• Volume: 10
• Category: Article
• ISSN: 0951-4198
• DOI: 10.1002/(sici)1097-0231(199611)10:14<1802::aid-rcm761>3.0.co;2-y

✦ Synopsis

Curie

-point pyrolysis/triple quadrupole mass spectrometry (F'y/MS/MS) has been used with tetramethylammonium hydroxide (TMAH) to conduct in sifu methylation of nucleic acid bases. Nitrogen bases in free nucleotides, oligonucleotides, calf thymus DNA and whole bacterial cells reacted in sifu during pyrolysis with TMAH to form the methylated bases. Derivatization increased the volatility of the nitrogen bases and the mass of the diagnostic base peaks, thereby removing them from the positions of lower-mass background peaks. The degree of methylation as a function of TMAH concentration for the oligonucleotide, calf thymus DNA, and the whole bacteria samples was determined and found to correlate with the nature of DNA. The methylated bases were identified by their positive-ion electron ionization fragmentation patterns and confirmed with tandem mass spectrometry. The detection of the methylated bases by Py/MS/MS facilitates the goal of identifying the nucleic acids in a complex mixture (i.e. whole bacterial cells) without extraction and prior derivatization.

Instrumental

An Extrel (Pittsburgh, PA, USA) ELQ-400 triple quadrupole mass spectrometer fitted with a Curie-point pyrolysis unit was used for the direct analyses of all samples.