A nano-chip-LC/MSn based strategy for characterization of modified nucleosides using reduced porous graphitic carbon as a stationary phase.

LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nano-chip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 µ × 150 mm, 5 µ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MS( n ) identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh(5)C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh(5)C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh(5)C was readily identified by its MH(+) m/z 260 and MS(n) fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips.

Identification of 5-hydroxycytidine at position 2501 concludes characterization of modified nucleotides in E. coli 23S rRNA.

Complete characterization of a biomolecule's chemical structure is crucial in the full understanding of the relations between their structure and function. The dominating components in ribosomes are ribosomal RNAs (rRNAs), and the entire rRNA-but a single modified nucleoside at position 2501 in 23S rRNA-has previously been characterized in the bacterium Escherichia coli. Despite a first report nearly 20 years ago, the chemical nature of the modification at position 2501 has remained elusive, and attempts to isolate it have so far been unsuccessful. We unambiguously identify this last unknown modification as 5-hydroxycytidine-a novel modification in RNA. Identification of 5-hydroxycytidine was completed by liquid chromatography under nonoxidizing conditions using a graphitized carbon stationary phase in combination with ion trap tandem mass spectrometry and by comparing the fragmentation behavior of the natural nucleoside with that of a chemically synthesized ditto. Furthermore, we show that 5-hydroxycytidine is also present in the equivalent position of 23S rRNA from the bacterium Deinococcus radiodurans. Given the unstable nature of 5-hydroxycytidine, this modification might be found in other RNAs when applying the proper analytical conditions as described here.