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

Abstract

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.

Time of acquisition and high spatial resolution mass spectrometry imaging

“…Although higher spatial resolution provides a greater depth of information, should it apply to every tissue analysis? For instance, while ultraviolet (UV) matrix‐assisted laser desorption/ionization (MALDI) has the capability of achieving subcellular resolution, many studies intentionally use a 50–100 μm pixel size to balance spatial resolution with the area of the region of interest (ROI), time of acquisition, and data file size 7–9 …”

“…For instance, while ultraviolet (UV) matrix-assisted laser desorption/ionization (MALDI) has the capability of achieving subcellular resolution, many studies intentionally use a 50-100 μm pixel size to balance spatial resolution with the area of the region of interest (ROI), time of acquisition, and data file size. [7][8][9] Given a defined ROI with constant dimensions, a higher spatial resolution yields an increased number of scans during an analysis, which translates into an increase in both the time of acquisition and data file size. For this reason, it is common to increase the pixel size for analysis of larger specimens or ROI; pixel sizes as high as 250 μm have been reported to allow for reasonable data collection and analysis times.…”

FT-ICR Mass Spectrometry Imaging at Extreme Mass Resolving Power Using a Dynamically Harmonized ICR Cell with 1ω or 2ω Detection

“…The KPMP Consortium (Veličković et al) and Tressler et al. improved MALDI MSI data after factorial design optimization of the deposited matrix using an automatic sprayer on mice’s kidney tissue sections. , In the presented work, the amount of deposited MALDI matrix was investigated in terms of signal suppression for the analytes of interest when varying the number of laser shots per pixel (see Figure S2). For this purpose, serial sagittal mouse brain slices were prepared with varying amounts of sprayed α-HCCA matrix of 10 and 5 nmol per mm 2 , respectively.…”

“…Different experimental parameters were evaluated extensively in the literature including slice thickness, 16 − 18 matrix and solvent selection, 19 − 23 and optimization of the automatized matrix deposition. 24 When using the recommended parameters and optimized methods intended for the previous ICR design, the “Infinity Cell”, the best performance in terms of R.P. FWHM and mass accuracy was far from instrument specifications due to abnormally large mass shifts.…”

“…The sample preparation affects the ionization efficiency as well as the local diffusion of the analytes in tissue sections. Different experimental parameters were evaluated extensively in the literature including slice thickness, − matrix and solvent selection, − and optimization of the automatized matrix deposition . When using the recommended parameters and optimized methods intended for the previous ICR design, the “Infinity Cell”, the best performance in terms of R.P.…”