Label‐free surface‐enhanced Raman spectroscopy of lipid‐rafts from hepatocyte plasma membranes

Abstract

Lipid rafts are sphingomyelin/cholesterol-rich domains present in the plasma membrane of eukaryotic cells. In the hepatocyte, it has been shown that these domains intervene and modify a wide range of functions from which cell signaling in health and disease is of major importance. The present investigation proposes a novel strategy for the study of plasma membrane lipid rafts using surface-enhanced Raman spectroscopy (SERS) and spectra processing employing the principal component analysis (PCA) in correlation with conventional biochemical techniques. SERS has been used for artificial membranes; our approach has the advantage that allows studying purified biological membranes using small volumes of biological samples and a very simple protocol. Therefore, the use of SERS/PCA represents an important advantage for the study not only of the structure and composition of biological membranes but also for the understanding of the functions carried by the membrane-embedded proteins. Our work using SERS/PCA for the first time shows a correlation when studying the composition of lipid rafts from the cell plasma membrane and the catalytic activity of the Ca 2+ -ATPase, and its direct association with the presence of specific lipids located in membrane lipid rafts. Also, we show that changes in plasma membrane and the catalytic activity of the Ca 2+ -ATPase activity present in the plasma membrane of hepatocytes after partial depletion of membrane cholesterol carried out by methyl-beta-cyclodextrin correlate with the changes in the enzyme activity and the presence of cholesterol-rich domains of lipid rafts.

Normal vibrations of ternary DOPC/DPPC/cholesterol lipid bilayers by low-frequency Raman spectroscopy

“…5,6 To explore membrane properties, bilayers made from synthetic lipids are oen used. [1][2][3][4]7 The heterogeneity of model membranes was studied with NMR and EPR, 1,2,[8][9][10] small-angle neutron scattering, 11,12 uorescence microscopy, 13,14 Raman scattering, [15][16][17][18][19][20] computer simulation, 21,22 and other techniques.…”

“…Vibrational frequencies of specic functional groups obtained in Raman scattering experiment [15][16][17][18][19][20] are sensitive to the local environment of lipids and proteins, providing so information on the lipid order. Recently, low-frequency Raman scattering spectra 23,24 were found to be capable for detection of THz acoustic vibrations of the bilayer as a whole.…”

Spectroscopy of model-membrane liposome-protein systems: complementarity of linear dichroism, circular dichroism, fluorescence and SERS

“…The incubation of liposomes with metal nanoparticles [77][78][79][80] is simple and does not require much preparation, with quality of data largely being dependent on the quality of the nanoparticles. The method has proved to be useful for lipid membrane analysis [81].…”

Recent advances in linear and nonlinear Raman spectroscopy. Part XII

“…Lipid rafts are sphingomyelin/cholesterol‐rich domains present in the plasma membrane of eukaryotic cells. They conclude that the use of SERS/PCA represents an important advantage for the study, not only of the structure and composition of biological membranes but also for the understanding of the functions carried by the membrane‐embedded proteins . Dina et al reported the discrimination of haloarchaeal genera using Raman spectroscopy and robust methods for multivariate data analysis.…”

“…They conclude that the use of SERS/PCA represents an important advantage for the study, not only of the structure and composition of biological membranes but also for the understanding of the functions carried by the membrane-embedded proteins. [53] Dina et al reported the discrimination of haloarchaeal genera using Raman spectroscopy and robust methods for multivariate data analysis. They aimed at developing a rapid and nondestructive method to predict the genus-level taxonomic affiliation of phenotypically similar halophilic Archaea of Halobacteria class.…”