Novel Ambient Liquid Extraction-Based Mass Spectrometry Imaging Techniques For Lipidomes

Lipids are a class of important endogenous metabolites.They can provide energy for organisms but also participate in cell signal transduction and regulate cell growth,differentiation,aging and other processes.Therefore,studying the spatial distribution of lipids in biological samples is of great significance for further revealing the metabolic activities of living organisms.Mass spectrometry imaging(MSI)is a sensitive,in situ and multiplexed approach for the characterization and localization of molecules in samples.Although mass spectrometry imaging techniques has been developed rapidly in recent years,there are still two problems: 1.accurate absolute quantitation for MSI.The main factor limiting the quantitative abilities of MSI is the dependence of the analyte ion signals on the tissue’s physicochemical and morphological properties,which is mainly attributed to the tissue-specific sampling/extraction efficiencies;2.The high detection coverage for low abundance/low responsive endogenous compounds are still challenging.low detection coverage in mass spectrometry imaging is mainly attributed to the strong ion suppression from high abundance/high responsive lipids(such as glycerophospholipids)to low abundance/low responsive lipids(such as glycolipids or glycerides).In order to solve the above problems,the main research contents of this paper are as follows:(1)A single-probe-based ambient liquid extraction system was established according to previous publications,and coupled it with an electrospray ionization-ion trap(IT)-time-of-flight(TOF)mass spectrometer to construct a new mass spectrometry imaging platform,which the spatial resolution was determined to be 200 μm.The conditions were systematically optimized in sampling,such as extraction solvent,flow rate,scan time of sample and sampling time in one pixel.It was found that higher LMJ stability and higher detection coverage were obtained with the new system compared with conventional system.Finally,a total of 127 lipids were detected,including 91 glycerophospholipids,13 glycerides,13 sphingolipids,10 free fatty acids and 19 small metabolites.By comparing the imaging results of endogenous lipids with the Singles-probe MSI and Flow-probe MSI,we found that higher spatial resolution and less hot spots/noise were obtained with the former imaging probe.(2)To achieve per-pixel absolute quantitation in MSI of endogenous lipids,the mass transfer kinetics of endogenous lipids in the ambient liquid extraction were investigated for the first time.The results demonstrated that the mass transfer kinetics of endogenous lipids were varied between different lipid species and between different brain regions,and standard addition in the tissue could not compensate for the variations.For quantitation of the endogenous lipids in single imaging pixel,the mass transfer kinetic model was proposed,and was used for prediction of the original amount of lipids in the tissue by fitting the experimental extraction kinetic curves in each pixel to the kinetic model.The experimental data was demonstrated to be well fitted to the model,and the prediction of original concentrations of PCs and CBs in the tissue proved to be accurate(relative errors <23%)with extraction time down to18 s.The new quantitation method was demonstrated to have better reproducibility and wider linear range,and be able to provide more distinct spatial distribution and more accurate quantitation results for qMSI than those with method without kinetic model prediction.Moreover,the accuracy of absolute quantitation results was verified by quantitative LC–MS method.(3)A new tissue imprinting-in situ liquid extraction MSI method was developed to address the narrow coverage range of MSI.Porous graphitic carbon(PGC)materials have selective adsorption to different kinds of lipids under different solvent environments.For example,lipids with polar hydrophilic heads have higher adsorption efficiency in aprotic solvents,while higher adsorption efficiency for non-polar lipids such as glycerides in protic solvents.Based on this phenomenon,PGC were made into imprinting-plate to perform imprinting extraction preserved the spatial distribution of the tissue,so as to enrich the low abundance/low responsive glycerides and remove high abundance/high responsive phospholipids.Then,using the above-mentioned extraction technology for scanning desorption imaging.First,the optimal spray solution for imprinting extraction and the optimal desorption solution for in-situ liquid extraction was determined through the results of standard adsorption experiments under different solvent conditions.Then,the imprinting efficiency(about 2-fold)was improved with the assistance of electrospray during mass spectrometry imaging of tissue imprinting.And finally,a total of 58 low-abundance and low-response lipids in rat cerebellum were detected,including 32 diglycerides,10 sphingolipids(4sphingomyelin,6 ceramides),which provides a 2-4 times greater coverage than direct tissue imaging methods.By comparing the MSI of these lipids with traditional direct tissue imaging results,we found that the signal of some lipids was improved 10-fold in MSI,and the distribution of glycerides and sphingolipids was less disturbed by the phospholipid signal.These results all indicate that the single-probe liquid extraction mass spectrometry imaging technology based on tissue imprinting technology can achieve mass spectrometry imaging of lowabundance and low-response lipids with wider coverage and less interference.