Demonstrating voxel-by-voxel (V × V) single-point calibration in liver tissue by IR-MALDESI quantitative MSI

Quantitative mass spectrometry imaging (qMSI) provides information regarding the colocalization, relative abundance, and concentration of a target analyte in a tissue without homogenization. Ionization sources, including IR-MALDESI, commonly utilize an on-tissue spatial calibration curve approach; however, this approach has several limitations including tedious sample preparation, and this approach does not account for local matrix effects. To compensate for these two limitations, we developed voxel-by-voxel (V × V) quantification to provide an internal standard calibration point for every voxel which requires a simple sample preparation and accounts for local matrix effects. In this work, we evaluate the performance of V × V quantification against the spatial calibration curve to assess the quantitative capacity of this newly developed method. Quantification of glutathione (GSH) on a per-voxel basis involves homogenously spraying a known amount of stable isotope-labeled glutathione (SIL-GSH) on a microscope slide. Next, we mount liver sections on top of the coated slides and image them using IR-MALDESI MSI. Statistical analysis demonstrated high precision for V × V quantification over a wide concentration range; however, the method's accuracy is currently limited due to the sprayer's configuration. Results support the feasibility of V × V quantification as evidenced by concentration heatmaps. Additionally, V × V quantification allows for parallel reaction monitoring (PRM) imaging which provides high specificity. Combined with relativity, straightforward sample preparation, and promising initial statistics, the V × V method offers significant advantages over spatial calibration curves.