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New approach for epitranscriptomics

Quantifying RNA methylations by LC-MS/MS

Dr Alexandre David, Amandine Amalric, Aurore Attina, Pr Sylvain Lehmann, Dr Jerome Vialaret, Pr Christophe Hirtz, Clinical Proteomics Platform, IRMB-PPC, INM, Univ Montpellier, CHU Montpellier, INSERM CNRS, Montpellier, France

Dr Alexandre David

Amandine Amalric

Aurore Attina

Pr Sylvain Lehmann

Dr Jerome Vialaret

Pr Christophe Hirtz

Clinical Proteomics Platform, IRMB-PPC, INM, Univ Montpellier, CHU Montpellier, INSERM CNRS, Montpellier, France

Epitranscriptomics is an emerging and promising research field covering biochemical RNA modifications within a cell. It comprises more than 170 RNA chemical marks, including RNA methylation which can regulate the expression of defined genes, and it is present in all type of RNA, such as messenger RNA (mRNA).

Today, mass spectrometry coupled to a liquid chromatography (LC-MS/MS) has become a method of choice to comprehensively measure modified nucleosides from a biological sample. This application note presents a methodology for quantifying methylated nucleosides from cellular mRNA using LC-MS/MS (Shimadzu LCMS-8060).

Introduction

Epitranscriptomics investigates modifications to RNA, which is involved in the production of proteins. This branch of research makes it easier to understand the connection between RNA modifications and diseases. For example, the epitranscriptome is modified in many tumors when compared to healthy cells.

Why is epitranscriptomics so promising?

There is a high number of RNA modifications with more than 170 RNA chemical marks described so far, including RNA methylation
These modifications are present in all RNA species (mRNA, tRNA, rRNA, ncRNA and so on)
RNA modifications are involved in all steps of post-transcriptional regulation of gene expression: RNA structure, splicing, export; stability, cellular localization and translation
they are involved in key cellular functions such as survival, growth and differentiation.

The N6-methyladenosine nucleoside (m⁶A) – the most common and abundant modifications on RNA in eukaryotes (i.e. organisms whose cells have a nucleus) – is a striking example. This modification occurs not only on mRNA but also on noncoding RNA (rRNA, tRNA, snRNA). As m⁶A is important for mRNA regulation in normal physiological condition, any dysregulation of its level can lead to disease. [1]

Current technologies to detect m⁶A modification [2] use next-generation sequencing techniques and deliver highly valuable information in term of m⁶A mapping at the genome-wide level. However, they cannot provide precise quantitative results. Today, mass spectrometry coupled to a liquid chromatography (LC-MS/MS) has become the method of choice for accurately quantifying modified nucleosides from biological samples. This application note presents a method for precise quantification of methylated nucleosides from cellular mRNA using LC-MS/MS (Shimadzu LCMS-8060).

LC analysis conditions

System

Nexera LC-40

Separation column

Synergi Fusion-RP C18 column (250 mm x 2 mm, 4 µm, 80 Å)

Mobile phase

A: 5 mM ammonium acetate, adjusted to pH 5.3 with acetic acid
B: acetonitrile

Gradient elution

Time (min)	[A] (%)	[B] (%)
0.0	100	0
3.0	100	0
3.1	99	1
13.0	92	8
23.0	60	40
25.0	100	0
30.0	100	0

Flow rate

0.4 mL/min

Oven temperature

35 °C

Autosampler temperature

4 °C

Injection volume

5 μL

MS analysis conditions

System

Triple Quadrupole LCMS-8060

Ionization method

ESI (positive)

Nebulizing gas flow

2.5 L/min

Drying gas flow

3 L/min

Heating gas flow

12.5 L/min

Interface temperature

325 °C

DL temperature

225 °C

Heat block temperature

380 °C

Table 1: Analytical conditions

Molecules	Transitions	Retention time (min)	Target Dwell Time (msec)	Target Collision Energy (E)	Target Q1 Pre Bias (V)	Target Q3 Pre Bias (V)	Interface Voltage (kV)
Adenosine (A)	268.0 > 136.0	11.8	197.0	-18.0	-24.0	-24.0	1.0
2’O-methyladenosine (A_m)	282.0 > 136.0	14.6	397.0	-17.0	-14.0	-24.0	1.5
N1-methyladenosine (m¹A)	282.1 > 150.1	5.4	197.0	-21.0	-12.0	-26.0	1.5
N6-methyladenosine (m⁶A)	282.1 > 150.1	15.9	397.0	-20.0	-12.0	-16.0	1.5
N6,N6-dimethyladenosine (m^6,6A)	296.0 > 164.1	18.3	530.0	-25.0	-22.0	-20.0	1.5
N6,2’O-dimethyladenosine (m⁶A_m)	296.0 > 150.0	17.6	397.0	-15.0	-18.0	-32.0	1.5
Cytidine (C)	244.1 > 112.0	3.8	197.0	-12.0	-17.0	-29.0	1.0
2’O-Methycytidine (C_m)	258.1 > 112.0	7.9	142.0	-15.0	-18.0	-32.0	1.5
N3-methylcytidine (m³C)	258.1 > 126.0	4.5	197.0	-13.0	-10.0	-14.0	1.5
N5-methylcytidine (m⁵C)	258.0 > 126.0	7.3	157.0	-17.0	-14.0	-25.0	1.0
Guanosine (G)	284.1 > 152.0	8.6	142.0	-15.0	-24.0	-23.0	1.5
2’O-methylguanosine (G_m)	298.1 > 152.0	10.9	174.0	-12.0	-12.0	-17.0	1.5
N1-methylguanosine (m¹G)	298.1 > 166.0	10.6	142.0	-15.0	-11.0	-18.0	0.5
N7-ethylguanosine (m⁷G)	298.3 > 166.0	7.3	157.0	-10.0	-30.0	-18.0	1.5
N2,N7-dimethylguanosine (m^2,7G)	312.1 > 180.0	10.3	142.0	-12.0	-12.0	-17.0	2.5
N2,N2,N7-trimethylguanosine (m^2,2,7G)	326.15 > 194	12.4	197.0	-20.0	-15.0	-21.0	1.5
Uridine (U)	245.1 > 113.0	5.2	197.0	-11.0	-28.0	-26.0	1.0
2’O-methyluridine (U_m)	259.1 > 113.0	9.5	142.0	-9.0	-11.0	-19.0	2.5

Table 2: MRM parameters

lw_dark — Figure 1: Workflow for label-free quantification of RNA methylations using LCMS-8060

Pre-analytical steps

Experimental workflow for quantification of methylated nucleosides by LC-MS/MS is shown in figure 1.

10⁶ cells CTC44 (Circulating Tumor Cells) were seeded into 150 mm x 15 mm petri dishes and cultured for 4 days at 37 °C under humidified 5 % CO₂ in high glucose DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 2 mM glutamine and 10 % FCS (Fetal Calf Serum).

RNA extraction was achieved using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. mRNA was isolated twice using an GenElute mRNA purification kit (Sigma). First, 400 ng of RNA (in 20 µL final) was digested by 5 units of RNA 5’ pyrophosphohydrolase (New England Biolabs) for 2 h at 37 °C with NEB buffer. Then decapped RNA was digested into nucleotides by 1 unit of nuclease P1 (Penicillium citrinum, Sigma) for 2 h at 42 °C on NH₄OAc buffer (10 mM, pH 5.3). Finally, nucleotides were dephosphorylated into nucleosides using 1 unit of alkaline phosphatase (Escherichia coli, Sigma) for 2 h at 37 °C on NH₄OAc buffer (100 mM). [3]

After adding 60 µL of phase A (ammonium acetate, pH 5.3), the samples are filtered (0.22 μm pore size, 4 mm diameter, Millipore), and 5 μL of the solution was injected into LC-MS/MS.

Analytical conditions

For the measurement of nucleosides using LC-MS/MS, the nucleosides are separated by reverse phase UHPLC (ultra-performance liquid chromatography) and detected by the Shimadzu LCMS-8060 triple-quadrupole mass spectrometer in multiple reactions monitoring (MRM) positive electrospray ionization (ESI) mode. Details of analysis conditions and MRM parameters are given in tables 1 and 2.

Modomics Database [4] contains information of MRM transitions of numerous modified nucleosides, and enables simultaneous measurement of several methylated nucleosides.

Quantification of methylated nucleosides from messenger RNA (mRNA)

The quantification of methylated nucleosides from mRNA yielded results for ten nucleoside species from the target panel of 14 RNA methylations (figure 3). The remaining four species have only been observed in rRNA, tRNA, and snRNA, and are – to the author’s knowledge – not present in mRNA (figure 2).

Furthermore, for most of the methylations detected, a lower signal is observed for mRNA compared to total RNA (figure 4). For example, m¹A is found in mRNA, which accounts for only 5 % of the total cell RNA, and also in tRNA and rRNA, which respectively represent 15 % and 80 %.

In the case of mRNA purification, the ion signal was enhanced for methylations that are mostly predominantly found in mRNA, such as m⁶A and m⁶A_m (figures 4 and 5).

Analytical conditions

Modomics Database [4] contains information of MRM transitions of numerous modified nucleosides, and enables simultaneous measurement of several methylated nucleosides.

Quantification of methylated nucleosides from messenger RNA (mRNA)

In the case of mRNA purification, the ion signal was enhanced for methylations that are mostly predominantly found in mRNA, such as m⁶A and m⁶A_m (figures 4 and 5).

Conclusion

RNA methylations label free quantification using a triple quadrupole LC-MS/MS system (LCMS-8060 mass spectrometer coupled to Nexera LC-40 in MRM mode) represents a sensitive and reliable approach for epitranscriptomics studies. This application can be implemented for a large panel of biological samples (and RNA species). It is a straightforward and readily applicable experimental setup.

Literature