Poster abstracts

Poster number 57 submitted by Whitney Houser

Enzymatic generation of 3’-phosphate from 2’,3’-cyclic phosphate endonuclease digestion products of recombinant RNase U2 for improved LC/MS analysis of RNAs

Whitney M. Houser (Chemistry Department, University of Cincinnati), Annika B. Kotter (University of Antwerp), Balasubrahmanym Addepalli (Chemistry Department, University of Cincinnati), Patrick Limbach (Chemistry Department, University of Cincinnati)

Abstract:
De novo sequencing of RNAs using LC-MS involves RNA mass mapping, which employs the use of endoribonucleases to digest the nucleic acid strand into detectable oligonucleotides. Ribonuclease U2 cleaves at the 3’ end of purine bases, but occasionally leaves a 2’,3’-cyclic phosphate at the 3’ end of its digestion products. This complicates mass spectra, resulting in overlapping peaks, lowered peak intensities, and reduced CID scans. An enzymatic approach for reducing the amount of these cyclic phosphate-ending digestion products is used. Bacteriophage lambda protein phosphatase (LPP) is used as a post-RNase U2 step in order to hydrolyze cyclic phosphate bonds, and for labelling purposes, as a means of introducing the label into the sample. These digestion products are then separated using reverse-phase HPLC, and analyzed using ESI-MS/MS. A dramatic reduction in the number of cyclic phosphate-ending digestion products is observed after treatment with LPP. Additionally, LPP is shown to be capable of introducing 18O label into oligoribonucleotides via digestion in the presence of heavy oxygen-containing water, allowing for the extension of current CARD methods to include digestion products ending in adenosines.

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Keywords: Mass spectrometry, Enzymatic Digestion, RNA sequencing