Optimized MALDI2-Mass\nSpectrometry Imaging for Stable\nIsotope Tracing of Tissue-Specific Metabolic Pathways in Mice

Spatial stable isotope tracing metabolic\nimaging is a cutting-edge\ntechnique designed to investigate tissue-specific metabolic functions\nand heterogeneity. Traditional matrix-assisted laser desorption ionization-mass\nspectrometry imaging (MALDI-MSI) techniques often struggle with low\ncoverage of low-molecular-weight (LMW) metabolites, which are often\ncrucial for spatial metabolic studies. To address this, we developed\na high-coverage spatial isotope tracing metabolic method that incorporates\noptimized matrix selection, sample preparation protocols, and enhanced\npost-ionization (MALDI2) techniques. We employed this approach to\nmouse kidney, brain, and breast tumors to visualize the spatial dynamics\nof metabolic flow. Our results revealed diverse regional distributions\nof nine labeled intermediates derived from <sup>13</sup>C<sub>6</sub>-glucose across glycolysis, glycogen metabolism, and the tricarboxylic\nacid (TCA) cycle in kidney tissues. In brain sections, we successfully\nmapped six intermediates from the TCA cycle and glutamate-glutamine\n(Glu-Gln) cycle simultaneously in distinct neurological regions. Furthermore,\nin breast cancer tumor tissues, our approach facilitated the mapping\nof nine metabolic intermediates in multiple pathways, including glycolysis,\nthe pentose phosphate pathway (PPP), and the TCA cycle, illustrating\nmetabolic heterogeneity within the tumor microenvironment. This methodology\nenhances metabolite coverage, enabling more comprehensive imaging\nof isotope-labeled metabolites and opening new avenues for exploring\nthe metabolic landscape in various biological contexts.