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.