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Nucleic Acids Research, 2002, Vol. 30, No. 23 e135
© 2002 Oxford University Press

Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry

Jonas Mengel-Jørgensen and Finn Kirpekar*

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Campusvej 55, 5230 Odense M, Denmark

*To whom correspondence should be addressed. Tel: +45 6550 2414; Fax: +45 6593 2781; Email: f.kir{at}bmb.sdu.dk

Mass spectrometry plays a central role in the characterisation of modified nucleotides, but pseudouridine is a mass-silent post-transcriptional modification and hence not detectable by direct mass spectrometric analysis. We show by the use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry that pseudouridines in tRNA can be specifically cyanoethylated by acrylonitrile without affecting the uridines. The tRNA was cyanoethylated and then subjected to digestion with either RNase A or RNase T1. Cyanoethylated digestion fragments were identified by mass spectrometric comparison of untreated and acrylonitrile-treated samples, where the addition of one acrylonitrile resulted in a mass increment of 53.0 Da. The exact modified nucleotide could be identified by tandem mass spectrometry on the cyanoethylated digestion fragment. The methodology was used to identify additional one 4-thiouridine and one pseudouridine in tRNATyrII from Escherichia coli. Furthermore, we observed that RNase A is highly tolerant towards nucleotide modifications, only being inhibited by 2'-O-methylation, whereas RNase T1 cleavage is affected by most nucleotide modifications.


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