Acidic Methanol Treatment Facilitates Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging of Energy Metabolism

Abstract

Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [¹³C₆]nicotinamide coupled to MALDI-MSI ("Iso-imaging") in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [¹³C₆]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.

Figure 1

Treatment of tissue section with MeOH + 0.05% FA maintains the histological morphology, denatures protein, removes lipids and highly polar metabolites, and improves the detection of phosphate-containing energy metabolites. (a) H&E images and MALDI images of selected metabolites (dTTP, dATP, ATP, ADP-ribose) before and after wash with 5 mL of MeOH + 0.05% FA demonstrate the histological morphology is maintained and metabolites are not moving. MALDI experiment was performed with a raster size of 20 μm. Anatomical regions are denoted as (i) deep cerebral nuclei, (ii) white matter, (iii) granule cell layer, and (iv) molecular layer. Scale bar = 500 μm. (b) Quantification of protein from serial sections of a kidney using bicinchoninic acid (BCA) assay. Running quantified protein on a native gel reveals no total protein content change (n = 3 tissue per condition) before and after wash with MeOH + 0.05% FA. While proteins are not being washed away, Coomassie blue staining of protein lysates from control and washed tissue sections separated by Native PAGE shows that lysates from tissues washed with methanol displayed increased mobility, and the effect was augmented by the inclusion of formic acid (FA), suggesting that proteins from washed samples are denatured. (c) Heatmap of selected compounds across four different wash conditions (log₂ fold of change): no wash control, 5 mL of chloroform, 5 mL of MeOH, 5 mL of MeOH + 0.05% FA. Compounds marked with * are detected in positive mode with a THAP matrix, while all other compounds are detected in negative mode with an NEDC matrix. Chloroform removes lipids and increases signals for polar metabolites. MeOH also removes lipids and selected highly polar metabolites such as taurine, glutamine, and glutamate while improving the signal of other polar metabolites. MeOH + 0.05% FA removes lipids and highly polar metabolites while enhancing the detection of phosphate-containing energy metabolites.

Figure 2

Acidic methanol treatment enables the detection of ADP, ATP, NAD⁺, NADH, NADP⁺, and NADPH in positive mode, revealing relative ATP and ADP signal across mouse kidney, as well as NAD⁺ and NADH synthesis from [¹³C₆]nicotinamide infusion. (a) Signal of ADP, ATP, NAD⁺, NADH, NADP⁺, and NADPH from mouse kidney before and after wash with 5 mL of MeOH + 0.05% FA, using four different matrices: CHCA, DHB, super-DHB, and THAP. (b) Upon infusion with [¹³C₆]nicotinamide (NAM), NAD⁺, and NADH get labeled, and the [¹³C₆] labeling ratio shows a pattern that distinguishes blood vessels (i), medulla (ii), outer stripe (iii), and cortex (iv) (n = 3 technical replicates). ATP relative to ADP raw signal (calculated from raw ion counts, thus not the actual concentration ratio) varies across different regions. Statistical analyses were performed using GraphPad Prism software. **p value < 0.05; ***p value < 0.005, ****p value < 0.0005. Scale bar 2 mm. (c) Schematic of the NAD⁺ synthesis pathway from [¹³C₆]nicotinamide (NAM). NAD⁺ then converts to NADH through a reduction reaction. (d) Immunofluorescence of 4′,6-diamidino-2-phenylindole (DAPI, as a control) and NMNAT3 in kidney cross section shows that NMNAT3 expression is highest in cortex region. Scale bar: 1 mm.

Figure 3

Differential glycolytic labeling in skeletal muscle fiber types is revealed by short glucose infusion and pulse-chase infusion coupled with an acidic methanol wash. (a) Schematic of isotope tracing experiments performed to determine the source of glycolytic intermediates in muscle. The panel on the left depicts a 2.5-hour infusion of [U-¹³C]glucose to visualize the muscle regions that use glucose directly via labeling in glycolytic intermediate FBP in the muscle (left) and the panel on the right pulse-chase injection to visualize the muscle regions that use glycogen (right). (b) Schematic of anticipated histological features after cryosectioning the muscle. A typical cross-section captures soleus (composed of Type 1 and Type 2a fibers), plantaris (mixed fiber), and gastrocnemius muscles containing mostly Type 2b muscle fibers. (S: soleus, P: plantaris and G: gastrocnemius) (c–e) Serial sections of mouse leg muscles from 2.5 h [U-¹³C] glucose infusion. (c) Immunohistochemistry to identify muscle fiber type in legs of mice. Red staining indicates fibers positive for anti-MYH7, a marker of type 1 fibers. Green staining indicates fibers positive for anti-MYH2a, a marker of type 2a fibers. Blue staining indicates fibers positive for anti-MYH2b, a marker of Type 2b fibers. (d, e) Metabolite images from short glucose infusion in skeletal muscle. FBP labeling from glucose and NADH levels are distinctly high in the soleus, rich in type 1 muscle fibers as revealed by wash. NADH levels are low in the gastrocnemius. Scale bar: 0.5 mm. (f−h) Serial sections of mouse leg muscles from pulse-chase glucose infusion. (f) Immunohistochemistry to identify muscle fiber type in legs of mice. Red staining indicates fibers positive for anti-MYH7, a marker of type 1 fibers. Green staining indicates fibers positive for anti-MYH2a, a marker of Type 2a fibers. Blue staining indicates fibers positive for anti-MYH2b, a marker of Type 2b fibers. (g, h) Metabolite images from pulse-chase glucose infusion in skeletal muscle. FBP labeling was seen across the tissue regardless of fiber type, indicating glycogen breakdown. Scale bar: 0.5 mm.