Per-pixel absolute quantitation for mass spectrometry imaging of endogenous lipidomes by model prediction of mass transfer kinetics in single-probe-based ambient liquid extraction

Abstract

With the development of mass spectrometry imaging (MSI), techniques providing quantitative information on the spatial distribution have attracted more attentions recent years. However, for MSI of endogenous compounds in bio-samples, the uncertainty of locally varied sampling efficiencies always hinders accurate absolute quantitation. Here single-probe was used for ambient liquid extraction MSI in rat cerebellum, and standards of phosphatidylcholines (PCs) and cerebrosides (CBs) were doped in extraction solvent. The extraction kinetic curves of endogenous lipids in the ambient liquid extraction during probe parking in single pixel of tissue were investigated. From the results, the extraction kinetic curves were varied between different lipid species in different brain regions, resulting in variations of extraction efficiencies between imaging pixels, and calibration with standards deposited in tissue could not compensate for the variations. In our approach, the theoretical kinetic model of ambient liquid extraction was established, and original concentrations of endogenous lipids in each pixel of tissue were predicted by fitting the experimental extraction kinetic curve in each imaging pixel to the model. The experimental data was demonstrated to be well fitted to the kinetic model with R² > 0.86, and only with 18-s extraction in each pixel, the original lipid concentrations were predicted accurately with relative errors <23%. With the new method, totally 157 lipids and small metabolites were imaged, and per-pixel quantitation was achieved for 19 PCs and 4 CBs. Compared with conventional quantitative MSI (q-MSI) method, the new q-MSI method had better reproducibility and wider linear range, and produced better contrast in the quantitative images of lipids in brain tissue with less hot spots and noises. The absolute quantitation results by the new method were verified by quantitative LC-MS method with Pearson'r > 0.9 and the slope of the linear fitting line of the correlation plot near 1.

Keywords: Ambient liquid extraction techniques; Mass transfer kinetic model; Quantitative mass spectrometry imaging; Single-probe.