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

RNA
L Peter SarinSebastian A Leidel

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. Fur...Continue Reading

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Citations

Mar 21, 2019·Biological Chemistry·Paria Asadi-AtoiStefanie Kellner
Feb 24, 2021·Critical Reviews in Biochemistry and Molecular Biology·Yasemin YoluçStefanie Kellner
Feb 19, 2021·Journal of Pharmaceutical and Biomedical Analysis·Takahiro KogakiKazutake Tsujikawa
Apr 4, 2021·Genes·Manuel KöstersSebastian A Leidel
Apr 22, 2021·Science China. Life Sciences·Jing LiRu-Juan Liu
Dec 5, 2018·Biochemistry·Leticia Pollo-Oliveira, Valérie de Crécy-Lagard
Oct 23, 2019·Analytical Chemistry·Mark S LowenthalKaren W Phinney

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