Jun 29, 2026
Approach to spatially profile free modifed nucleic acid metabolites through DNA RNA Elements Areal Mass Spectrometry (DREAMS)
- Yan Peng1,
- Liang Xue1,
- Magdalena Koziol1
- 1Chinese Institute for Brain Research, Beijing (CIBR)
Protocol Citation: Yan Peng, Liang Xue, Magdalena Koziol 2026. Approach to spatially profile free modifed nucleic acid metabolites through DNA RNA Elements Areal Mass Spectrometry (DREAMS). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoj54bg4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 22, 2026
Last Modified: June 29, 2026
Protocol Integer ID: 319540
Keywords: nucleic acid metabolite, modified nucleic acid metabolite, dna rna elements areal mass spectrometry, free modifed nucleic acid, metabolite, biological sample, moving biological sample
Abstract
DNA RNA Elements Areal Mass Spectrometry (DREAMS) entails directing a spray solution at a moving biological sample to release metabolites. The liberated modified nucleic acid metabolites are then detected by MS and spatially visualized for comprehensive sample analysis.
Experimental animals
3 of knockout or disease mice were used as the experimental group and 3 of C57BL/6J wild-type mice were used as the control group.
Obtaining the brains of the mice in each group
Anesthetize the mice by intraperitoneal injection of 2.5% avertin at a dose of 0.35 g/kg.
After sacrifice, take the brains of the mice, including olfactory bulb to medulla regions.
Place on ice and wash with pre-cooled phosphate-buffered saline.
Carefully suck the remaining phosphate-buffered saline on the surface of the brain samples with dust-free paper.
Place the mouse brains in cell culture dishes.
Store in a -80°C freezer for later use.
Preparing brain sections
Take out the mouse brains obtained above; place in the chamber of the freezing microtome at -22 °C for 20 minutes.
Cut the left and right sides of the brains with a knife and fix them on the sample holder.
Cut sections with a thickness of 20 microns per section, along the sagittal direction or coronal direction.
Mount the sections on positively charged glass slides. One glass slide was applied with 6 brain sections from 6 mice (1-3 from the experimental group and 4-6 from the control group).
Two more slides were applied in the same order, marked and stored in a -80°C freezer.
DREAMS approach
Take out the slides from the -80 °C freezer before the MSI experiment.
Dry at room temperature for 20 minutes.
For the MSI system, it was set to have nitrogen pressure of 0.63-0.67 MPa and spray flow rate of 5 μL/min.
Spray solution is 80% acetonitrile in water.
The angle between the needle and the slide was 57° and the height therebetween was 36.5 mm.
The horizontal x-direction scanning rate was set to be 0.2 mm/s.
The y-direction vertical interval was set to be 0.1 mm.
For the mass spectrometer connected, the duration of the detection process was equal to the movement time of the MSI sample platform.
The temperature of the ion transfer tube was set to be 350°C, and the resolution ratio was set to be 1.2 million.
Full positive or negative scanning was performed, with the scanning mass-to-charge ratio range of 78-780.
The target of AGC was set to be 1E7.
The maximum injection time was set to be 100 ms, to obtain the mass spectrometry data.
Spatial quantification of nucleic acid metabolites
Convert the data to CDF format using Xcalibur software.
Process using MassImager software to subtract the background, with the mass tolerance being set to 10 ppm.
The nucleic acid metabolites comprised the metabolites of RNA and/or DNA, such as free modified nucleosides, free nucleosides, free modified nucleotides and free nucleotides.
The image obtained through m/z 303.2330 ion channel (which corresponds to arachidonic acid) selected from the MSI data is acquired in a negative ion mode.
14 brain regions were selected based on the schematic diagram of the brain structure of the sagittal plane using MassImager software, including olfactory bulb, pons and medulla, hippocampus, midbrain, cerebellum, cerebral cortex, anterior olfactory nucleus, caudate putamen, thalamus, hypothalamus, basal forebrain, ventral striatum, fornix and corpus callosum.
Then the MSI data of these 14 brain regions were extracted by using MassImager software.
The changed free modified nucleosides, free nucleosides, free modified nucleotides and free nucleotides were found in various brain regions of the mice according to the programed MSI data analysis procedure.
Automatically create Excel files for storing the mass spectrometry data of all brain regions of interest.
Select the ion intensities according to the monitoring ions of the targets.
Integrate the data in all Excel files.
Normalize the data with at least three technological or biological replicates.
Compare the data of the mice from the experimental and control groups, and perform differential analysis of the content of each target free modified nucleoside, free nucleoside, free modified nucleotide or free nucleotide in these 14 brain regions one by one, to generate box plots.