42] Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry

Abstract

Publisher Summary This chapter discusses preparation and enzymatic hydrolysis of Deoxyribo-nucleic acid (DNA) and Ribo Nucleic Acid (RNA) for mass spectrometry. The utilization of mass spectrometry for direct analysis of DNA and RNA enzymatic digests places certain restrictions on the types of buffers and salts tolerated in the hydrolysate. While thermospray liquid chromatography-mass spectrometry (LC/MS) is relatively insensitive to their presence, when a fast atom bombardment (FAB)-based method is used, there is already substantial background from the matrix alone, and reduction of any additional interferences from the digest can only simplify the analysis. For an electron ionization (EI)-based method, in which the dried digest is trimethylsilylated, the presence of ammonium sulfate, Mg 2+, Tris-Cl, etc., utilized in typical digestion protocols will attenuate yields of derivatized nucleosides. Thymidine and cytidine are most severely affected, and removal of salts following digestion is recommended. Isolated DNA or RNA typically contains varying amounts of the other nucleic acid, and while traces of RNA in DNA (and vice versa) generally do not confound the biological experiment, they will be readily apparent in the mass spectrometric analysis, and have the potential to interfere in the analysis.