Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

Abstract

In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC–MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding 13C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single modifications in weighable quantities, this SIL-IS consists of a nucleoside mixture covering all detectable RNA modifications of Escherichia coli, yet in small and initially unknown quantities. For absolute in addition to relative quantification, those quantities were determined by a combination of external calibration and sample spiking of the biosynthetic SIL-IS. For each nucleoside, we thus obtained a very robust relative response factor, which permits direct conversion of the MS signal to absolute amounts of substance. The application of the validated SIL-IS allowed highly precise quantification with standard deviations <2% during a 12-week period, and a linear dynamic range that was extended by two orders of magnitude.

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

“…The method also extends the linear dynamic range for quantification by two orders-of-magnitude by equalizing ion suppression effects due to saturation (gray area in Fig. 4) (Kellner, Ochel, et al, 2014). …”

“…Note the extended dynamic range in the gray area (saturation range). Adapted and modified from (Gu, Begley, & Dedon, 2014; Kellner, Ochel, et al, 2014; Su et al, 2014)…”

Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs

“…LC-MS/MS analysis of RNA from mouse ES cells and Drosophila S2 cells (cultured under standard conditions) was performed as described previously (Kellner et al 2014). Further details are provided in Supplemental Methods.…”

RNA 5-Methylcytosine Analysis by Bisulfite Sequencing

“…Recent developments, especially the application of sensitive mass spectrometry tools to nucleic acid chemistry (i.e., liquid chromatography combined with mass spectrometry, LC-MS) has considerably increased analytical sensitivity, opening up possibilities for quantifying also the modifications status of low-abundance RNAs (Kellner et al, 2014;Li et al, 2008;Russell & Limbach, 2013). Using LC-MS has provided unprecedented insights into the dynamic landscape of RNA modifications (including m 5 C) in abundant RNAs, especially during stress conditions (Chan et al, 2010(Chan et al, , 2012.…”