Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection.
A protocol that utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide (CMC) derivatization to detect the post-transcriptionally modified nucleoside, pseudouridine, in RNA has been optimized for RNase digests. Because pseudouridine is mass-silent (i.e., the mass of pseudouridine is the same as the mass of uridine), after CMC-derivatization and alkaline treatment, all pseudouridine residues exhibit a mass shift of 252 Da that allows its presence to be easily detected by mass spectrometry. This protocol is illustrated by the direct MALDI-MS identification of pseudouridines within Escherichia coli tRNA(TyrII) starting from microgram amounts of sample. During this optimization study, it was discovered that the post-transcriptionally modified nucleoside 2-methylthio-N(6)-isopentenyladenosine, which is present in bacterial tRNAs, also retains a CMC unit after derivatization and incubation with base. Thus, care must be exercised when applying this MALDI-based CMC-derivatization approach for pseudouridine detection to samples containing transfer RNAs to minimize the misidentification of pseudouridine.
Durairaj
Anita
A
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221-0172, United States.
Limbach
Patrick A
PA
D004926:Escherichia coli / Q000235:genetics; D005092:Exonucleases / Q000378:metabolism; D007094:Imides / Q000737:chemistry*; D009025:Morpholines / Q000737:chemistry*; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry; D011560:Pseudouridine / Q000737:chemistry*; D012313:RNA / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D007094:Imides; D009025:Morpholines; C527904:N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide; D009841:Oligonucleotides; D011560:Pseudouridine; D012313:RNA; D005092:Exonucleases; D006163:Ribonuclease T1