Factorial Design to Optimize Matrix Spraying Parameters for MALDI Mass Spectrometry Imaging

Matrix deposition is a critical step in obtaining reproducible and spatially representative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging data. To date, few limited studies have examined the optimization of matrix spraying parameters for maximizing analyte extraction while minimizing analyte delocalization. Herein, we present a study using automated pneumatic spraying with a heated sample-holder tray to determine an optimized model for mouse whole kidney lipid imaging using a 2,5-dihydroxybenzoic acid matrix in which the solvent flow rate, nozzle velocity, and sample heating were optimized using a two-level factorial experimental design. Parameters examined to determine the optimum model include the number of analytes, the matrix crystal size, off tissue delocalization, the signal intensity, and spray time. Our results show that sample heating using a heated tray while spraying improves the MALDI imaging performance. This improvement is possible because higher solvent flow rates can be used in the pneumatic sprayer, allowing for better sample extraction, while sample delocalization is minimized due to sample heating.