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.

Ribosomal RNA from all organisms contains post-transcriptionally modified nucleotides whose function is far from clear. To gain insight into the molecular interactions of modified nucleotides, we investigated the modification status of Thermus thermophilus 5 S and 23 S ribosomal RNA by mass spectrometry and chemical derivatization/primer extension. A total of eleven modified nucleotides was found in 23 S rRNA, of which eight were singly methylated nucleotides and three were pseudouridines. These modified nucleotides were mapped into the published three-dimensional ribosome structure. Seven of the modified nucleotides located to domain IV, and four modified nucleotides located to domain V of the 23 S rRNA. All posttranscriptionally modified nucleotides map in the center of the ribosome, and none of them are in contact with ribosomal proteins. All except one of the modified nucleotides were found in secondary structure elements of the 23 S ribosomal RNA that contact either 16 S ribosomal RNA or transfer RNA, with five of these nucleotides physically involved in intermolecular RNA-RNA bridges. These findings strongly suggest that the posttranscriptional modifications play a role in modulating intermolecular RNA-RNA contacts, which is the first suggestion on a specific function of endogenous ribosomal RNA modifications.All cellular protein synthesis is performed by ribosomes, which are large ribonucleoprotein particles. The prokaryote ribosome consists of two stable and separable entities, a 50 S and a 30 S subunit. The 50 S subunit contains two rRNAs of ϳ3000 and 120 nucleotides (23 S and 5 S rRNA, respectively) and around 35 proteins, whereas the 30 S subunit contains 16 S rRNA of ϳ1600 nucleotides and around 20 proteins; the exact numbers vary with the species. Eukaryotic ribosomes are larger, but structural features are remarkably conserved between the domains of life.rRNAs are post-transcriptionally modified at specific nucleotides, but the number of modified nucleotides varies greatly. The large ribosomal RNA in mitochondria contains just a few modified nucleotides (1), Escherichia coli 23 S rRNA has 25 (2, 3), whereas over 100 are found in vertebrate cytoplasmic 28 S rRNA (4). The function of post-transcriptional rRNA modifications is far from clear, although they have been implicated in various processes. Specific rRNA methylation is used by numerous antibiotics-producing microorganisms as a means of autoprotection (see e.g. Refs. 5 and 6), but only a small fraction of post-transcriptional modifications can be assigned to this well defined function. E. coli 23 S rRNA modifications cluster in functionally principal parts of the ribosome such as the peptidyl transferase center and inter-subunit bridges when modeled into the structure of the Haloarcula marismortui large ribosomal subunit (7). The modified nucleotides in the central domains of 23 S rRNA from H. marismortui itself are all located in regions of intra-or intermolecular RNA-RNA contact (8) suggesting structure stabilization.The post-transcriptionall...

One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.

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