Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells

Abstract

Exploration of the epitranscriptome requires the development of highly sensitive and accurate technologies in order to elucidate the contributions of the more than 100 RNA modifications to cell processes. A highly sensitive and accurate ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously detect and quantify 28 modified and four major nucleosides in less than 20 min. Absolute concentrations were calculated using extinction coefficients of each of the RNA modifications studied. A comprehensive RNA modifications database of UV profiles and extinction coefficient is reported within a 2.3-5.2 % relative standard deviation. Excellent linearity was observed 0.99227-0.99999 and limit of detection values ranged from 63.75 attomoles to 1.21 femtomoles. The analytical performance was evaluated by analyzing RNA modifications from 100 ng of RNA from human pluripotent stem cell-derived neural cells. Modifications were detected at concentrations four orders of magnitude lower than the corresponding parental nucleosides, and as low as 23.01 femtograms, 64.09 attomoles. Direct and global quantitative analysis of RNA modifications are among the advantages of this new approach.

Figure 1.

Averaged UV-spectra of major and modified nucleosides. X-axis is represented by the wavelength range from 190 to 340 nm. The Y-axis is in absorbance units. The absorbance at 260 nm is indicated by a vertical line. Nucleoside concentrations were calculated by measuring absorbance at 260 nm and using extinction coefficients calculated at pH 3.5 (Table 1). Standard deviation from multiple scans and experiments is represented by the gray shadow (Further details can be found in the Supplementary Information).

Figure 2.

Extracted ion chromatogram of the four the major nucleosides, A, G, U and C and 28 modified nucleoside standards. The one-dimensional separation is shown on the left (A). The full names of the nucleosides are found in Table 1 with the following peak identification: 1, C; 2, D; 3, Ψ; 4, cmnm⁵U; 5, m¹A; 6, s²U; 7, m⁵C; 8, mnm⁵s²U; 9, m¹acp³Ψ; 10, U; 11, s²C; 12, m⁷G; 13, Cm; 14, m⁶U; 15, I; 16, m⁵U; IS, [¹³C]₁₀,[¹⁵N]₅G; 17, G; 18, f⁵C; 19, mo⁵U; 20, s⁴U; 21, A; 22, Um; 23, Gm; 24, m²G; 25, m¹I; 26, mcm⁵U; 27, m¹G; 28, ac⁴C; 29, m⁶A; 30, mo⁵ s²U; 31, mcm⁵s²U; 32, i⁶A. To ilustrate the resolution achieved for separation of the nucleosides in the time and mass dimmensions, the period of 8 to 12 min (shadowed area) is represented in a 3D plot (B). The time axis is the X-axis, molecular ion mass [MH⁺] is the Y-axis and the product [BH₂⁺] mass ions are on the Z-axis. Each of the four color representation is a canonical parental nucleoside and/or its corresponding modifications eluting during 8–12 min.

Figure 3.

Experimental workflow. Graphical description of the step-by-step method for the preparation and UHPLC–MS analysis of nucleosides from total RNA extracted from cerebral cortex cells differentiated in culture. Human stem cells were cultured for 77 days and total RNA extracted, purified and readily hydrolyzed to nucleoside via a two-step enzymatic hydrolysis prior to composition analysis using UHPLC–MS/MS.

Figure 4.

Absolute concentrations of RNA major and modified nucleosides during neural stem cell differentiation in culture. Day 0 (A), 19 (B), 33 (C), 49 (D) and 77 (E). The order of the nucleosides at Day 0 (A) is represented in a descending order of concentration. The Y-axis and the order of the X-axis were maintained in plots representing Day 19–77 (B–E). Dynamic range of concentrations is shown by the absolute values represented on respective tables that are inserted. The tables show that there is a 10⁴-fold concentration difference between the major and modified nucleosides. Error bars represent the standard deviation of five instrument replicates.