SpotOn: A Tool to Enhance Echo Acoustic Standard Deposition for Quantitative Mass Spectrometry Imaging

The quantitative evaluation of therapeutics in the drug discovery pipeline is essential for elucidating their pharmacokinetic and pharmacodynamic profiles within complex biological systems. Traditionally, autoradiography and liquid chromatography-mass spectrometry (LC-MS) have been employed for such analyses. However, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is emerging as a promising alternative, offering a more rapid untargeted workflow. Quantitative MALDI-MSI (qMSI) relies on accurate calibration to achieve robust and reproducible quantification of analytes. Here, we present an open-source software tool, SpotOn, designed to facilitate the discrete deposition of low-volume calibration standards onto glass slides or tissue sections. By enabling precise placement and significantly reducing the volume of standards required, our tool affords the generation of more calibration points across a wider dynamic range, thereby improving the robustness and accuracy of quantitative analyses. Software was validated using JQ1 on both glass slides and murine liver tissue. The resulting calibration curves demonstrated strong linearity, with coefficients of determination (R ²) exceeding 0.92. This tool streamlines the qMSI workflow, conserves valuable reagents, and enhances analytical reliability, making it a helpful resource.