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

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      J   Am Soc Mass Spectrom. 2011 Jul;22(7):1242-51. doi:   10.1007/s13361-011-0126-8. Epub 2011 Apr 15.


      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.

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