Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry.
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 tRNA(TyrII) 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.
Mengel-Jørgensen
Jonas
J
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Campusvej 55, 5230 Odense M, Denmark.
Kirpekar
Finn
F
D000181:Acrylonitrile / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D011560:Pseudouridine / Q000032:analysis* / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000032:analysis; D012365:RNA, Transfer, Tyr / Q000032:analysis; D006163:Ribonuclease T1 / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014529:Uridine / Q000737:chemistry; D015003:Yeasts / Q000235:genetics
D012360:RNA, Transfer, Phe; D012365:RNA, Transfer, Tyr; D011560:Pseudouridine; D012343:RNA, Transfer; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic; D000181:Acrylonitrile; D014529:Uridine