Nano LC-MS using capillary columns enables accurate quantification of modified ribonucleosides at low femtomol levels

Abstract

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

“…This approach provides reproducible and reliable relative quantification of global nucleoside modification changes in various organisms. Furthermore, we also show that our method is directly compatible with previously published spike-in [ 15 N] stable isotope labeling techniques for signal normalization 17,23 , which further increase the quantification accuracy. This UPLC-MS method can also be used for dynamic monitoring of tRNA modification changes via pulse-chase labelling approaches, such as NAIL-MS 16 .…”

“…The quality and purity of isolated tRNA was assessed by denaturing electrophoresis on 10% polyacrylamide gel (Supplementary Data). Dephosphorylated mononucleosides for MS analysis were generated as previously described 23 . Prior to MS analysis, the completeness of cleavage was verified by gel electrophoresis.…”

“…Figure S7). This approach significantly improves the accuracy of relative quantification by eliminating the variance that stems from the differences in ribonucleoside ionization efficiency 23 . However, metabolic labeling is not without its limitations.…”

“…is the prevailing approach used by numerous laboratories 4,[9][10][11][12][13][14][15][16][17][18][19][20] . In addition, bespoke methods have been developed using other stationary phases, such as HILIC 21,22 and porous graphitic carbon (PGC) 23,24 .…”

Modulation of Escherichia coli Translation by the Specific Inactivation of tRNAGly Under Oxidative Stress

“…The detailed protocol for the analysis of RNA by mass spectrometry has been described elsewhere (Sarin et al, 2018). In brief, tRNAs were digested to single nucleosides essentially, as previously described (Alings et al, 2015).…”