RRC ID 53623
Author Houser WM, Butterer A, Addepalli B, Limbach PA.
Title Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography-mass spectrometry.
Journal Anal Biochem
Abstract Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2',3'-cyclic phosphates and 3'-linear phosphates. To generate a homogeneous 3'-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3'-phosphate-containing products that could be characterized by LC-MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce (18)O labels within the 3'-phosphate of digestion products when incubated in the presence of H2(18)O, allowing prior isotope labeling methods for mass spectrometry to include digestion products from RNase U2.
Volume 478
Pages 52-8
Published 2015-6-1
DOI 10.1016/j.ab.2015.03.016
PII S0003-2697(15)00121-9
PMID 25797349
MeSH Bacteriophage lambda / enzymology* Bacteriophage lambda / genetics Base Sequence Chromatography, Liquid / methods Clostridium thermocellum / enzymology* Clostridium thermocellum / genetics Codon Endoribonucleases / genetics Endoribonucleases / metabolism* Escherichia coli / genetics Escherichia coli / metabolism* Genes, Synthetic Molecular Sequence Data Phosphoric Diester Hydrolases / genetics Phosphoric Diester Hydrolases / metabolism* RNA, Bacterial / chemistry RNA, Bacterial / metabolism* Recombinant Proteins / genetics Recombinant Proteins / metabolism Tandem Mass Spectrometry / methods
IF 2.877
Times Cited 12
Prokaryotes E. coli