Characterization of freeze-fractured epithelial plasma membranes on nanometer scale with ToF-SIMS

• Published in: Analytical and bioanalytical chemistry 2015-03, Vol.407 (8), p.2203-2211
• Main Authors: Draude, Felix, Körsgen, Martin, Pelster, Andreas, Schwerdtle, Tanja, Müthing, Johannes, Arlinghaus, Heinrich F.
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Asymmetry; Biochemistry; Cell culture; Cell Line, Tumor; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell membranes; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Choline; Diseases; Epithelial cells; Epithelial Cells - chemistry; Epithelial Cells - metabolism; Food Science; Freeze Fracturing; Humans; Investigations; Ion beams; Laboratory Medicine; Lipid Bilayers - chemistry; Lipid Metabolism; Lipids; Lipids - chemistry; Mass spectrometry; Mass Spectrometry Imaging; Membranes; Metabolism; Methods; Monitoring/Environmental Analysis; Monolayers; Nanostructure; Physiological aspects; Plasma; Research Paper; Scientific imaging; Secondary ion mass spectrometry; Silicon; Silicon substrates; Silicon wafers; Spectrometry, Mass, Secondary Ion - methods; Time-of-flight mass spectrometry; Wafers
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-014-8334-2

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to characterize the freeze-fracturing process of human epithelial PANC-1 and UROtsa cells. For this purpose, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine standard samples were investigated to find specific signals with both high specificity and signal intensity. The results were used to investigate single cells of subconfluent cell layers prepared with a special silicon wafer sandwich preparation technique. This freeze-fracturing technique strips cell membranes off the cells, isolating them on opposing silicon wafer substrates. Criteria were found for defining regions with stripped off cell membranes and, on the opposing wafer, complementary regions with the remaining cells. Measured ethanolamine/choline and serine/choline ratios in these regions clearly showed that in the freeze-fracturing process, the lipid bilayer of the plasma membrane is split along its central zone. Accordingly, only the outer lipid monolayer is stripped off the cell, while the inner lipid monolayer remains attached to the cell on the opposing wafer, thus allowing detailed analysis of a single lipid monolayer. Furthermore, it could be shown that using different washing procedures did not influence the transmembrane lipid distribution. Under optimized preparation conditions, it became feasible to detect lipids with a lateral resolution of approximately 100 nm. The data indicate that ToF-SIMS would be a very useful technique to study with very high lateral resolution changes in lipid composition caused, for example, by lipid storage diseases or pharmaceuticals that interfere with the lipid metabolism.