Using immobilized enzymes to reduce RNase contamination in RNase mapping of transfer RNAs by mass spectrometry.
RNase (ribonuclease) mapping by nucleobase-specific endonucleases combined with mass spectrometry (MS) is a powerful analytical method for characterizing ribonucleic acids such as transfer RNAs. Typical free solution enzymatic digestion of RNA samples results in a significant amount of RNase being present in the sample solution analyzed by MS. In some cases, the RNase can lead to contamination of the high performance liquid chromatography and MS instrumentation. Here we investigate and compare several different approaches for reducing or eliminating contaminating RNase from the digested RNA sample before LC-MS analysis. Approaches using immobilized RNases were found to be most effective, with no enzyme carryover into the digested sample detected. Among the various options for immobilized RNases, we show that carbodiimide-based reactions can be used to couple RNases to carboxylic acid-terminated magnetic beads. The immobilized enzymes retain biological activity, are re-usable, and do not interfere with subsequent LC-MS analysis of the expected RNase digestion products. The use of immobilized RNases provides a simple approach for eliminating enzyme contamination in mass spectrometry-based RNase mapping experiments.
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, OH, 45221-0172, USA.
Annika
A
Butterer
Margret
M
Zorc
Colette M
CM
Castleberry
Patrick A
PA
Limbach
D001483:Base Sequence; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004800:Enzymes, Immobilized; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012343:RNA, Transfer; D012260:Ribonucleases