Shotgun analysis provides a quantitative snapshot of the lipidome composition of cells, tissues, or model organisms; however, it does not elucidate the spatial distribution of lipids. We demonstrate that shotgun analysis quantified low-picomole amounts of lipids isolated by laser capture microdissection (LCM) of hundred micrometer-sized histological zones visualized at the cryosections of tissues. In a first step, we identified metabolically distinct periportal (pp) and pericentral (pc) zones in mouse liver by immunostaining of 20 μm thick cryosections. LCM was used to ablate, catapult, and collect the tissue material from 10 to 20 individual zones covering a total area of 0.3−0.5 mm2 and containing ca. 500 cells. We quantified more than 200 lipid species from 17 lipid classes including glycero- and glycerophospholipids, sphingolipids, cholesterol esters, and cholesterol. Shotgun LCM revealed the overall commonality of the full lipidome composition of pp and pc zones along with significant (p < 0.001) difference in the relative abundance of 13 lipid species. Follow-up proteomics analyses of the same LCM obtained samples identified 13 known and 7 new protein markers exclusively present in pp or in pc zones and independently validated the specificity of their visualization, isolation, and histological assignment (Knittelfelder et al., 2018).
Our approach can be extended to other tissues with small adjustments to reveal the spatial distribution of lipids and proteins in defined regions of interest. Currently we are interested in the lipid composition of pancreatic islets from non-/diabetic patients as well as in liver zonation affected by NAFLD and tumor heterogeneity of hepatocellular cancer.
Liver specific LCM workflow. Serial cryo-sections were obtained and the middle one was subjected to immunostaining (glutamine synthetase to identify pericentral zones and DAPI to visualize nuclei dense bile ducts indicating periportal zones). Zones were matched to the unstained cryo-sections used for LCM. Collected material (0.3 – 0.5 mm2) was extracted for lipids and analyzed by shotgun lipidomics.
Knittelfelder, O. et al. (2018) ‘Shotgun Lipidomics Combined with Laser Capture Microdissection: A Tool to Analyze Histological Zones in Cryosections of Tissues’, Analytical Chemistry, 90(16), pp. 9868–9878. doi: 10.1021/acs.analchem.8b02004.