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. 2018 Oct;24(10):1403-1417.
doi: 10.1261/rna.065482.117. Epub 2018 Jul 16.

Nano LC-MS Using Capillary Columns Enables Accurate Quantification of Modified Ribonucleosides at Low Femtomol Levels

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Nano LC-MS Using Capillary Columns Enables Accurate Quantification of Modified Ribonucleosides at Low Femtomol Levels

L Peter Sarin et al. RNA. .
Free PMC article

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.

Keywords: mass spectrometry; nanoflow liquid chromatography; porous graphitic carbon; stable isotope labeling; transfer RNA modification.

Figures

FIGURE 1.
FIGURE 1.
Reliable ribonucleoside separation is achieved by reversed-phase chromatography using porous graphitic carbon (PGC) and C18 materials. (A) Representative chromatograms (A254-trace) obtained with PGC-A (left panel), PGC-B (middle panel), and C18 (right panel) separating 20 ribonucleoside standards (LSM, Supplemental Table 1). (B) Individual chromatograms obtained for each of the 20 ribonucleosides included in the LSM using run conditions as in A. Abbreviations follow the Modomics database convention (Boccaletto et al. 2018).
FIGURE 2.
FIGURE 2.
Ribonucleosides separation is robust and reproducible on PGC materials. Retention time and column durability analysis of 30 consecutive runs with pseudouridine (Ψ), 5-methylcytidine (m5C), inosine (I), adenosine (A), and 1-methylguanosine (m1G) on PGC-A and PGC-B columns. Representative chromatograms from runs 1, 5, 15, and 30 are shown. The table summarizes the retention time parameters for all tested nucleosides. Abbreviations follow the Modomics database convention (Boccaletto et al. 2018).
FIGURE 3.
FIGURE 3.
PGC and C18 capillaries enable the analysis of ribonucleosides in nLC ESI-MS/MS. (A) Representative total ion chromatograms (TIC) of 100 ng of the LSM or CSM (C18 only) analyzed on nanoflow capillary columns packed with PGC-A (left panel), PGC-B (middle panel), or C18 (right panel) material. (B) Extracted ion chromatograms (XIC) for adenosine (A), cytidine (C), guanosine (G), uridine (U), pseudouridine (Ψ), inosine (I), N4-acetylcytidine (ac4C), 1-methyladenosine (m1A), 2′-O-methyladenosine (Am), and N6-methyladenosine (m6A; CSM only). (C) Example MS1 spectra recorded at the retention times corresponding to the nucleosides analyzed in B. (NL) Normalized target level.
FIGURE 4.
FIGURE 4.
Comprehensive analysis of highly complex analytes is achieved using PGC-B. (A) Representative TIC chromatograms of 31 ribonucleoside standards (100 ng of CSM, Supplemental Table 1; left panels) and 250 ng of an enzymatic digest of bulk tRNA isolated from Saccharomyces cerevisiae strain BY4741 (right panels) analyzed on a nano PGC-B capillary column. (B) Overlay of the XICs for all CSM ribonucleosides detected in the respective samples. (C) XICs of selected nucleosides present in the samples. Note the capability of the PGC material to separate, with the exception of methylated guanosine, all positional isomers. (NL) Normalized target level.
FIGURE 5.
FIGURE 5.
Absolute quantification can be accomplished over a broad detection range. (A) Calibration curves for representative ribonucleosides analyzed on PGC-B (left panels) and C18 matrices (right panels) showing the observed XIC maximum intensity as a function of sample loaded (0.04–4000 pg). The error bars represent the standard deviation for each data point (n = 3). Linear regression (dark gray line) is used to determine the dynamic range of the instrument for each ribonucleoside on the respective matrix by observing the range at which a linear dependency between input amount and intensity is observed (vertical dashed bars indicate the range). (B) Summary of the linear quantification range determined for all 31 ribonucleosides present in the CSM (linear regression for all moieties is R2 ≥ 0.96). The matrix that provides the best overall performance (detection limit, quantification range, R2-value)—and is the recommended choice for analyzing the selected ribonucleoside—is highlighted in bold.
FIGURE 6.
FIGURE 6.
Accurate relative quantification of ribonucleosides can be achieved using stable isotope labeled internal spike-in standards. (A) XICs of adenosine in its native nonlabeled (left panel) form and with stable isotope (15N)-labeling (right panel, spike-in standard), as well as a schematic representation of the chemical structure of adenosine showing the 15N incorporation sites highlighted in gray. (B) MS1 spectrum for adenosine at RT 39.30 min. Note the increase in mass (∼5 Da) resulting from the incorporation of 15N in the base (15N-labeled mass in gray, nonlabeled mass in black). (C) MS2 fragmentation spectra of nonlabeled (top panel; m/z = 268.10) and 15N-labeled (bottom panel; m/z = 273.09) adenosine. Note the appearance of the base at m/z = 136.06 and m/z = 141.05, respectively, corresponding to the expected neutral loss of ribose (−132.04). (D) Cross-dilution series of nonlabeled and 15N-labeled enzymatic digests of bulk tRNA isolated from Chlamydomonas reinhardtii. Quantification of the maximum peak intensity of nonlabeled (solid dark gray line) versus 15N-labeled (solid light gray line) XICs of the canonical bases and representative ribonucleoside modifications. Shown is the abundance ratio of nonlabeled (N) and 15N-labeled (15N) maximum signal intensities (MaxI); Abundance = MaxI15N/(MaxI15N + MaxIN) or vice versa. The dotted lines represent the expected ratio of 15N-labeled (light gray) versus nonlabeled (dark gray) material present in the samples. (NL) Normalized target level.

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