Proteomics

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MS-based analysis of enzymatic methylation of an in vitro-transcribed lncRNA

ABSTRACT: The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we prepared two samples of an in vitro-transcribed yeast lncRNA (NME1, 340 nt long), one of which was treated with an RNA methyltransferase (NCL1) catalyzing the 5-methylcytidine (m5C) modification. These samples were subsequently digested with an RNA endonuclease (RNase) to generate oligonucleotide sequences of a length amenable to mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Hendrik Weisser  

LAB HEAD: Byron Andrews

PROVIDER: PXD016308 | Pride | 2020-03-31

REPOSITORIES: Pride

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Publications

A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry.

Wein Samuel S   Andrews Byron B   Sachsenberg Timo T   Santos-Rosa Helena H   Kohlbacher Oliver O   Kouzarides Tony T   Garcia Benjamin A BA   Weisser Hendrik H  

Nature communications 20200217 1

The field of epitranscriptomics continues to reveal how post-transcriptional modification of RNA affects a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. Exist  ...[more]

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