Quantitative Spatial Analysis of the Mouse Brain Lipidome by Pressurized Liquid Extraction Surface Analysis

Abstract

Here we describe a novel surface sampling technique termed pressurized liquid extraction surface analysis (PLESA), which in combination with a dedicated high-resolution shotgun lipidomics routine enables both quantification and in-depth structural characterization of molecular lipid species extracted directly from tissue sections. PLESA uses a sealed and pressurized sampling probe that enables the use of chloroform-containing extraction solvents for efficient in situ lipid microextraction with a spatial resolution of 400 μm. Quantification of lipid species is achieved by the inclusion of internal lipid standards in the extraction solvent. The analysis of lipid microextracts by nanoelectrospray ionization provides long-lasting ion spray which in conjunction with a hybrid ion trap-orbitrap mass spectrometer enables identification and quantification of molecular lipid species using a method with successive polarity shifting, high-resolution Fourier transform mass spectrometry (FTMS), and fragmentation analysis. We benchmarked the performance of the PLESA approach for in-depth lipidome analysis by comparing it to conventional lipid extraction of excised tissue homogenates and by mapping the spatial distribution and molar abundance of 170 molecular lipid species across different anatomical mouse brain regions.

Improved Extraction Repeatability and Spectral Reproducibility for Liquid Extraction Surface Analysis–Mass Spectrometry Using Superhydrophobic–Superhydrophilic Patterning

“…A number of groups have limited the spreading of the probe solvent by using electrodes 5 or applying pressure on top of the solvent. 4 Another way of limiting solvent spread is contact LESA 6 or performing laser ablation rather than liquid extraction. 7 It already has been observed that the spreading of the extraction solvent on hydrophilic surfaces is substantial 6 and that the use of nonpolar solvents such as chloroform dramatically increases the solvent spread.…”

Ambient Mass Spectrometry Imaging Using Direct Liquid Extraction Techniques

“…84 Such “contact” model LESA may be advantageous for the analysis of samples containing a protective layer on the surface. When installed on a fully automated robotic system, it enables high-throughput, spatially resolved analysis of metabolites, 85,86 lipids, 87–90 and proteins 91,92 in complex samples, including dried blood spots, 84,88 contact lenses, 89 tissues, 55,83,86,91 and microbial colonies. 84,93 LESA also has been used for analyzing molecules separated using TLC 83 and capillary electrophoresis.…”

“…87 In PLESA experiments, a pipet filled with ~500 nL of organic extraction solvent is pressed into the sample, forming a sealed microextraction volume. Improved extraction efficiency is achieved by applying headspace pressure to the pipet during the ~30 s extraction time.…”

“…PLESA has been used for spatial profiling of tissue sections with a spatial resolution of ~400 µm and a total footprint of ~800 µm left by the probe on the surface. 87 …”

“…The presence of lipids standards during extraction and ionization enabled quantification of 170 different lipid species from 11 lipid classes at each location. 87 The concentrations of the endogenous lipids were calculated using the known concentration of the internal standard of the lipid class and the intensity ratio of the endogenous lipid over the standard. 137 These experiments have demonstrated that the most abundant lipid class in all brain regions was PC, followed by phosphatidylethanolamine (PE).…”