Protocol for spatial metabolomics and isotope tracing in the mouse brain

Abstract

Mass spectrometry imaging enables high-resolution spatial chemical mapping, yet its application for dynamic analysis with tracers poses challenges. Here, we present a protocol for spatial metabolomics and isotope tracing in the mouse brain. We describe steps for tracer administration, tissue collection, and cryosectioning. We then detail procedures for matrix application, ion identification, and data analysis. This protocol delivers high-quality spatial metabolomics data and is well suited for region-specific tracing analysis in the brain.

Keywords: mass spectrometry; metabolism; neuroscience.

Graphical abstract

Figure 1

Tissue collection and preparation for sectioning (A) Dissection setup. (B) Measuring blood glucose before ¹³C-glucose injection. (C) Intraperitoneal injection of the ¹³C tracer. (D) Dissected brain illustration. (E) Separating brain hemispheres with a blade. (F) Midline region of the hemisphere after separation. (G) Hemisphere placement in the molding block. (H) Tissue freezing on dry ice.

Figure 2

Sectioning the brain tissue (A) Tissue placement in the cryostat chamber. (B) Application of OCT to the chuck. (C) Positioning the brain tissue laterally with the midline facing up. (D) Checking ITO slide conductivity. (E) Tissue sectioning. (F) Mounting tissue on the conductive side of the ITO slide. (G) Warming the tissue slightly with a finger to aid attachment. (H) Storing tissue in a 50 mL tube on dry ice before freezing at −80°C.

Figure 3

Matrix deposition (A) ITO slide before processing. (B and C) Loading the matrix into the matrix deposition boat for even distribution. (D) Placing the matrix and ITO slide side by side in the IMLayer. (E) Starting the matrix deposition process after preparing settings. (F) Checking the chamber temperature before opening to avoid burns. (G) ITO slide after matrix deposition. (H) Placing the slide in the appropriate cassette and (I) inserting it into the IMScope.

Figure 4

Analysis of MSI data (A) Metabolite distribution by signal intensity (left panel, red: high intensity, blue: low intensity), UMAP analysis (middle panel), segmentation distribution (right panel). (B) Region of interest of the cortex and cerebellum (left panel), UMAP analysis of cortex (top-middle panel) and cerebellum (bottom-middle panel) region, segmentation distribution of cortex (top-right panel) and cerebellum (bottom-right panel) region. (C) Chemical composition for each cluster of the entire brain (left panel), cortex (middle panel), and cerebellum (right panel). (D) Schematic of [U-¹³C]glucose metabolism and ¹³C tracer distribution across downstream metabolites. (E) Anatomical distribution of M0 (unlabeled) and M2 (labeled with two ¹³C carbons) of key metabolites derived from [U-¹³C]glucose, with enrichment analysis shown in the heatmap. (F) M0 and M2 isotopologue enrichment analysis of glutamate, glutamine, and GABA from MSI (N = 3 mice), as well as cerebellum and cortex extracts (N = 8) analyzed by GC-MS, from P21 mice administered the same [U-¹³C]glucose injection protocol. The Pearson correlation analysis (top-left panel, R = 0.86) between MSI and GC-MS is also illustrated, demonstrating the strong agreement between both methods. An unpaired Student’s t-test was used to perform statistical comparisons between groups. ∗p < 0.05, ∗∗p < 0.01.