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Ariadne: a database search engine for identification and chemical analysis of RNA using tandem mass spectrometry data.

Nakayama H, Akiyama M, Taoka M, Yamauchi Y, Nobe Y, Ishikawa H, Takahashi N, Isobe T - Nucleic Acids Res. (2009)

Bottom Line: Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values.The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts.It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

View Article: PubMed Central - PubMed

Affiliation: Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.

ABSTRACT
We present here a method to correlate tandem mass spectra of sample RNA nucleolytic fragments with an RNA nucleotide sequence in a DNA/RNA sequence database, thereby allowing tandem mass spectrometry (MS/MS)-based identification of RNA in biological samples. Ariadne, a unique web-based database search engine, identifies RNA by two probability-based evaluation steps of MS/MS data. In the first step, the software evaluates the matches between the masses of product ions generated by MS/MS of an RNase digest of sample RNA and those calculated from a candidate nucleotide sequence in a DNA/RNA sequence database, which then predicts the nucleotide sequences of these RNase fragments. In the second step, the candidate sequences are mapped for all RNA entries in the database, and each entry is scored for a function of occurrences of the candidate sequences to identify a particular RNA. Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values. The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts. It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

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Characterization of an unknown small RNA in yeast tRNA preparation. (a) Isolation of an unknown small RNA by anion-exchange chromatography. A mixture of yeast tRNA (tRNA typeX, Sigma R9001; 100 μg) was applied to a TSKgel DNA-NPR column (4.6 × 75 mm; Toso) and eluted with an 80-min gradient of NH4Cl (0.1–1.0 M) in 25 mM Tris–HCl buffer (pH 9.0) at a flow rate of 0.5 ml/min at 60°C. The fraction indicated by a double-headed arrow was collected, purified by reversed-phase chromatography (data not shown) and subjected to the LC–MS analysis after digestion with RNase T1 (see Materials and Methods section). (b) Base peak chromatogram of the RNase T1 digest of the small RNA.
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Figure 7: Characterization of an unknown small RNA in yeast tRNA preparation. (a) Isolation of an unknown small RNA by anion-exchange chromatography. A mixture of yeast tRNA (tRNA typeX, Sigma R9001; 100 μg) was applied to a TSKgel DNA-NPR column (4.6 × 75 mm; Toso) and eluted with an 80-min gradient of NH4Cl (0.1–1.0 M) in 25 mM Tris–HCl buffer (pH 9.0) at a flow rate of 0.5 ml/min at 60°C. The fraction indicated by a double-headed arrow was collected, purified by reversed-phase chromatography (data not shown) and subjected to the LC–MS analysis after digestion with RNase T1 (see Materials and Methods section). (b) Base peak chromatogram of the RNase T1 digest of the small RNA.

Mentions: During the purification of a commercial yeast tRNA preparation by anion-exchange chromatography, we found that an RNA component(s) eluted significantly later than the bulk of tRNAs (Figure 7a). This component had a greatly decreased mobility as compared with yeast tRNAs on polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (data not shown), suggesting that it might be an extra RNA component that was larger than typical yeast tRNAs. To examine whether Ariadne could identify this unknown RNA component, we purified the RNA by reversed-phase LC (Figure 7a) and digested it with RNase T1. The digest was then analyzed by nanoflow LC–MS/MS (Figure 7b), and the resulting MS/MS data were used to search against the genome database of S. cerevisiae. As shown in Figures 8 and 9, six genomic regions on yeast chromosome XII were identified based on their significantly high scores. These regions had nucleotide sequences in common with one another and contained eight RNase T1 fragments of the sample RNA identified by MS/MS ion search (Figure 10). The BLAST search of this nucleotide sequence against the yeast genome database clearly indicated that it encodes 5S rRNA, a 120-nt RNA that is slightly larger than typical tRNAs (which are about 75 nt). The MS/MS-based de novo sequencing of the major fragment of this RNA confirmed this conclusion (data not shown). The genome-sequencing study of Goffeau et al. (43) showed that the right arm of yeast chromosome XII contains approximately 140 tandem copies of a 9.1-kb sequence encoding rRNAs. However, the currently available S. cerevisiae genome database excludes most of the repeated sequence and contains only 1.1 Mb of the 2.4-Mb sequence of the total length of chromosome XII, including six copies of 5S rRNAs (43). It should be noted that the six chromosomal loci that were recognized by Ariadne include all of the genomic loci that encode 5S rRNA in the S. cerevisiae genome database. Thus, Ariadne could be useful for MS-based identification of unknown RNA components in biological mixtures.Figure 7.


Ariadne: a database search engine for identification and chemical analysis of RNA using tandem mass spectrometry data.

Nakayama H, Akiyama M, Taoka M, Yamauchi Y, Nobe Y, Ishikawa H, Takahashi N, Isobe T - Nucleic Acids Res. (2009)

Characterization of an unknown small RNA in yeast tRNA preparation. (a) Isolation of an unknown small RNA by anion-exchange chromatography. A mixture of yeast tRNA (tRNA typeX, Sigma R9001; 100 μg) was applied to a TSKgel DNA-NPR column (4.6 × 75 mm; Toso) and eluted with an 80-min gradient of NH4Cl (0.1–1.0 M) in 25 mM Tris–HCl buffer (pH 9.0) at a flow rate of 0.5 ml/min at 60°C. The fraction indicated by a double-headed arrow was collected, purified by reversed-phase chromatography (data not shown) and subjected to the LC–MS analysis after digestion with RNase T1 (see Materials and Methods section). (b) Base peak chromatogram of the RNase T1 digest of the small RNA.
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Figure 7: Characterization of an unknown small RNA in yeast tRNA preparation. (a) Isolation of an unknown small RNA by anion-exchange chromatography. A mixture of yeast tRNA (tRNA typeX, Sigma R9001; 100 μg) was applied to a TSKgel DNA-NPR column (4.6 × 75 mm; Toso) and eluted with an 80-min gradient of NH4Cl (0.1–1.0 M) in 25 mM Tris–HCl buffer (pH 9.0) at a flow rate of 0.5 ml/min at 60°C. The fraction indicated by a double-headed arrow was collected, purified by reversed-phase chromatography (data not shown) and subjected to the LC–MS analysis after digestion with RNase T1 (see Materials and Methods section). (b) Base peak chromatogram of the RNase T1 digest of the small RNA.
Mentions: During the purification of a commercial yeast tRNA preparation by anion-exchange chromatography, we found that an RNA component(s) eluted significantly later than the bulk of tRNAs (Figure 7a). This component had a greatly decreased mobility as compared with yeast tRNAs on polyacrylamide gel electrophoresis (PAGE) under denaturing conditions (data not shown), suggesting that it might be an extra RNA component that was larger than typical yeast tRNAs. To examine whether Ariadne could identify this unknown RNA component, we purified the RNA by reversed-phase LC (Figure 7a) and digested it with RNase T1. The digest was then analyzed by nanoflow LC–MS/MS (Figure 7b), and the resulting MS/MS data were used to search against the genome database of S. cerevisiae. As shown in Figures 8 and 9, six genomic regions on yeast chromosome XII were identified based on their significantly high scores. These regions had nucleotide sequences in common with one another and contained eight RNase T1 fragments of the sample RNA identified by MS/MS ion search (Figure 10). The BLAST search of this nucleotide sequence against the yeast genome database clearly indicated that it encodes 5S rRNA, a 120-nt RNA that is slightly larger than typical tRNAs (which are about 75 nt). The MS/MS-based de novo sequencing of the major fragment of this RNA confirmed this conclusion (data not shown). The genome-sequencing study of Goffeau et al. (43) showed that the right arm of yeast chromosome XII contains approximately 140 tandem copies of a 9.1-kb sequence encoding rRNAs. However, the currently available S. cerevisiae genome database excludes most of the repeated sequence and contains only 1.1 Mb of the 2.4-Mb sequence of the total length of chromosome XII, including six copies of 5S rRNAs (43). It should be noted that the six chromosomal loci that were recognized by Ariadne include all of the genomic loci that encode 5S rRNA in the S. cerevisiae genome database. Thus, Ariadne could be useful for MS-based identification of unknown RNA components in biological mixtures.Figure 7.

Bottom Line: Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values.The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts.It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

View Article: PubMed Central - PubMed

Affiliation: Biomolecular Characterization Team, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.

ABSTRACT
We present here a method to correlate tandem mass spectra of sample RNA nucleolytic fragments with an RNA nucleotide sequence in a DNA/RNA sequence database, thereby allowing tandem mass spectrometry (MS/MS)-based identification of RNA in biological samples. Ariadne, a unique web-based database search engine, identifies RNA by two probability-based evaluation steps of MS/MS data. In the first step, the software evaluates the matches between the masses of product ions generated by MS/MS of an RNase digest of sample RNA and those calculated from a candidate nucleotide sequence in a DNA/RNA sequence database, which then predicts the nucleotide sequences of these RNase fragments. In the second step, the candidate sequences are mapped for all RNA entries in the database, and each entry is scored for a function of occurrences of the candidate sequences to identify a particular RNA. Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values. The method was validated with MS/MS data of RNase T1 digests of in vitro transcripts. It was applied successfully to identify an unknown RNA component in a tRNA mixture and to analyze post-transcriptional modification in yeast tRNA(Phe-1).

Show MeSH