A Nano-Chip-LC/MSⁿ Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

Abstract

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 nanochip 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.

The Fish Pathogen Yersinia ruckeri Produces Holomycin and Uses an RNA Methyltransferase for Self-resistance

“…The digested RNA fragments were then submitted for HR-LC/ ESI/MS analysis. The following HPLC conditions were used for RNA fragment analysis according to the previous report (22): HYPERCARB column, Thermo Scientific, 100 ϫ 4.6 mm, 5 m; A: 50 mM formic acid in water; B: 50 mM formic acid in acetonitrile; gradient 0 -60 min 0 -100% B; 1 ml/min. High resolution electrospray ionization mass spectra were obtained using a Thermo Scientific MS system (LTQ XL/LTQ Orbitrap Discovery) coupled to a accela HPLC system (Thermo Scientific) (negative mode, capillary voltage Ϫ50 V, capillary temperature 320°C, auxiliary gas flow rate 10 -20 arbitrary units, sheath gas flow rate 40 -50 arbitrary units, spray voltage 4.5 kV, mass range 300 -3000 atomic mass units (maximum resolution 30,000)).…”

Recognition of guanosine by dissimilar tRNA methyltransferases

“…The procedures were based on previously published work (Crain 1990;Douthwaite and Kirpekar 2007;Giessing et al 2011). For MALDI-TOF mass spectrometry, each tRNA (1-2 pmol) was digested by RNase A (for EctRNA Leu transcripts) or by RNase T1 (for MjtRNA Cys transcripts) to completion, and the generated fragments were analyzed directly using 3-hydroxypicolinic acid as the matrix.…”

“…Mass spectra were recorded in positive ion mode with a reflectron Time-of-Flight mass analyzer on a PerSeptive Voyager-DE STR instrument (Applied Biosystems) (Douthwaite and Kirpekar 2007). LC-tandem mass spectrometry of tRNA nucleosides was performed according to a recently published procedure (Giessing et al 2011). After complete hydrolysis of the phosphodiester backbone (Crain 1990), tRNA nucleosides were separated on a porous graphitized carbon column with direct analysis on an ion trap mass spectrometer (Agilent XCT Ultra 6340) operated in positive ion mode.…”

Identification of 5-Hydroxycytidine at Position 2501 Concludes Characterization of Modified Nucleotides in E. coli 23S rRNA

“…Gradient separation was achieved using 0-90% acetonitrile with 1 mM oxalic acid to avoid nucleoside oxidation. 13 To obtain CID (collision-induced dissociation) fragment spectra in the low m/z range, we manually adjusted the fragmentation amplitude to 0.5 V for MS and to 0.6 V for MS n to secure a potential well deep enough to avoid ejection of fragment ions from the ion trap. 5-Hydroxycytidine standard was prepared enzymatically as recently described.…”