High-resolution ion mobility spectrometry-mass spectrometry of isomeric/isobaric ribonucleotide variants

Abstract

In this report, we explored the benefits of cyclic ion mobility (cIM) mass spectrometry in the analysis of isomeric post-transcriptional modifications of RNA. Standard methyl-cytidine samples were initially utilized to test the ability to correctly distinguish different structures sharing the same elemental composition and thus molecular mass. Analyzed individually, the analytes displayed characteristic arrival times (t_D ) determined by the different positions of the modifying methyl groups onto the common cytidine scaffold. Analyzed in mixture, the widths of the respective signals resulted in significant overlap that initially prevented their resolution on the t_D scale. The separation of the four isomers was achieved by increasing the number of passes through the cIM device, which enabled to fully differentiate the characteristic ion mobility behaviors associated with very subtle structural variations. The placement of the cIM device between the mass-selective quadrupole and the time-of-flight analyzer allowed us to perform gas-phase activation of each of these ion populations, which had been first isolated according to a common mass-to-charge ratio and then separated on the basis of different ion mobility behaviors. The observed fragmentation patterns confirmed the structures of the various isomers thus substantiating the benefits of complementing unique t_D information with specific fragmentation data to reach more stringent analyte identification. These capabilities were further tested by analyzing natural mono-nucleotide mixtures obtained by exonuclease digestion of total RNA extracts. In particular, the combination of cIM separation and post-mobility dissociation allowed us to establish the composition of methyl-cytidine and methyl-adenine components present in the entire transcriptome of HeLa cells. For this reason, we expect that this technique will benefit not only epitranscriptomic studies requiring the determination of identity and expression levels of RNA modifications, but also metabolomics investigations involving the analysis of natural extracts that may possibly contain subsets of isomeric/isobaric species.

Scheme 1.

Structures of the methyl-cytidine and methyl-adenosine monophosphate isomers examined in the study.

Scheme 2.

Schematic representation of the cyclic IM instrument employed in the study.

Figure 1.

IM-MS data obtained from canonical ribonucleotides on either a linear TW (left) or a cIM (right) device after a single pass.

Figure 2.

IM-MS analysis of individual methyl-cytidine standards (Scheme 1) and their equimolar mixture. Data obtained after 1 and 10 passes in the cIM device are respectively displayed on the left and right column (see Experimental).

Figure 3.

Multi-pass cIM analysis of methyl-cytidine mixture. After 10 passes, m⁵C and m⁴C were ejected from the separator, whereas the partially resolved Cm/m³C were submitted to additional passes to reach a total of a) 12, b) 20, c) 30, and d) 35 (see Experimental).

Figure 4.

Mass-selected time-resolved (MaSTeR) dissociation spectra obtained from the equimolar mixture of methyl-cytidine isomers. Panel a) shows the windows of t_D from which the various product ion spectra were obtained (see Experimental). In particular, panel b) corresponds to the population emerging at 74.4 ±0.5 ms; c) at 79.9 ±0.5 ms; d) at 78.2 ±0.5 ms; and e) at 76.0 ±0.5 ms. Precursor ions are marked with a red asterisk, whereas signals produced by base loss are marked with black circles or squares.

Figure 5.

Heatmap plots obtained by cIM after 3 passes (left) and linear TW (right) analysis of an exonuclease digestion mixture from total RNA extract of HeLa cells (see Experimental). The cIM separator fully resolved the uridine (U) and pseudo-uridine (Ψ) isomers, whereas the linear TW device displayed extensive overlap (insets).

Figure 6.

Multi-pass analysis of methyl-cytidine (a) and methyl-adenine (b) isomeric subsets present in the exonuclease mixture from total HeLa RNA (see Experimental). Isomeric sets with 336.06 and 360.07 m/z were isolated in the mass-selective Q, then analyzed after 10 (for methyl-cytidines) or 20 (for methyl-adenosines) passes in the cIM device.