Shotgun Lipidomics Combined with Laser Capture Microdissection: A Tool To Analyze Histological Zones in Cryosections of Tissues

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. Here we demonstrate
that shotgun analysis could quantify low-picomole amounts of lipids
isolated by laser capture microdissection (LCM) of hundred micrometer-sized
histological zones visualized at the cryosections of tissues. We identified
metabolically distinct periportal (pp) and pericentral (pc) zones
by immunostaining of 20 μm thick cryosections of a healthy mouse
liver. 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. Top-down shotgun profiling
relying upon computational stitching of 61 targeted selective ion
monitoring (t-SIM) spectra 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 pellets recovered from an aqueous phase saved after the
lipid extraction 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.