Demonstrating Voxel‐by‐Voxel (V × V) Single‐Point Calibration in Whole‐Body Zebrafish by IR‐MALDESI Quantitative MSI

This work describes a quantitative mass spectrometry imaging (qMSI) method comparison for the absolute quantification of arachidonic acid (AA) in whole-body zebrafish using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Whole-body zebrafish are structurally heterogeneous samples that are complex to analyze by many qMSI methods largely due to practical sample preparation limitations. However, the multi-organ quantification is valuable in zebrafish, especially for lipid-related investigations. In the standard workflow, the ion abundance of AA was normalized to a structural analogue that was sprayed on the slide before mounting the tissue. A series of calibration spots of stable isotope label (SIL) deuterated AA were spotted onto tissue to construct a calibration curve and subsequently calculate the concentration of endogenous AA in the tissue sections. The calculated values of AA using this method provided values significantly lower than the literature. In the subsequent workflow, the structural analogue was considered in a voxel-by-voxel (V × V) calculation of the concentration of AA in the tissues resulting in an AA concentration similar to the literature within whole-body zebrafish. The V × V method proved simpler in sample preparation, and more accurately quantified AA. This implies the potential utility of single-point V × V calibration for the qMSI of highly heterogeneous tissues.