Brief explanation of Ariadne
Accumulating evidence shows that diverse types of RNAs generated from non-coding (nc) regions of the genome play pivotal roles in a variety of cellular processes, such as chromatin remodeling, transcriptional regulation, precursor mRNA processing, gene silencing, centromere function and translational regulation.
Here, we present a novel method for automated identification of RNA by using tandem MS data from RNA nucleolytic fragments to search against a DNA/RNA sequence database. The method uses (i) nucleolytic fragmentation of sample RNA with an RNase, such as RNase T1, that has defined cleavage specificity, (ii) LC-MS/MS analysis of the fragments and (iii) analysis of the MS/MS dataset by Ariadne, a database search engine that we developed. We examined the performance of the method with a number of applications to identify mRNA synthesized in vitro or small RNA isolated from biological samples. We show here that Ariadne is the first genome-oriented search engine for RNA analysis that could be equivalent to the many sequence search engines that are widely used for proteomics, such as SEQUEST or Mascot.
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 experimental mass values are then compared with calculated oligonucleotide mass or product ion mass values, obtained by applying cleavage rules to the entries in a nucleotide sequence database. By using an appropriate scoring algorithm, the closest match or matches can be identified.
However, the identified nucleotides can belong more than one RNAs because variability of nucleotides is limited relative to the number of RNA species in a cell. For example, there can be only 1024 (=4*4*4*4*4) possible pentanucleotides although there should be more than 10^4 RNAs in human genome. The probability of co-existence of the nucleotides in an RNA
2. Search parameters for Ariadne
Ariadne receives search parameters including uninterpreted MS/MS data, a sequence database, and other parameters in text format from the search form. See details in 'Preparing the search' section of 'Ariadne Help' page.
Papers in which Ariadne is reported or used are listed below.
- 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 Apr;37(6):e47. Epub 2009 Mar 6. (link to publisher's site)
- An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes. Taoka M, Yamauchi Y, Nobe Y, Masaki S, Nakayama H, Ishikawa H, Takahashi N, Isobe T. Nucleic Acids Res. 2009 Nov;37(21):e140.(link to publisher's site)