A novel strategy for the comprehensive analysis of the biomolecular composition of isolated plasma membranes

• Published in: Molecular systems biology 2011-10, Vol.7 (1), p.541-n/a
• Main Authors: Thimiri Govinda Raj, Deepak B, Ghesquière, Bart, Tharkeshwar, Arun Kumar, Coen, Katrijn, Derua, Rita, Vanderschaeghe, Dieter, Rysman, Evelien, Bagadi, Murali, Baatsen, Pieter, De Strooper, Bart, Waelkens, Etienne, Borghs, Gustaaf, Callewaert, Nico, Swinnen, Johan, Gevaert, Kris, Annaert, Wim
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; Biomarkers; Cancer; Carbohydrates; Cations; Cell Line; Cell lines; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell surface; Cholesterol; Cholesterol - analysis; Cholesterol - metabolism; Composition; Convergence; Electrophoresis; EMBO20; EMBO31; Embryo fibroblasts; Embryos; Endosomes - genetics; Endosomes - pathology; eukaryotic cell systems; Fibroblasts; Gene Expression Profiling; Gene Knockout Techniques; Glycan; glycomics; Immunology; Intermediates; Lipid composition; lipidomics; Lipids; Lipogenesis; Magnetite Nanoparticles; Membrane Lipids - analysis; Membrane Lipids - metabolism; Membrane Proteins - analysis; Membrane Proteins - genetics; Membranes; Methodology; Methods; Mice; Nanoparticles; Pharmaceuticals; Phospholipids; Plasma; Plasma membranes; Presenilin 1; presenilin, proteomics; Presenilins - genetics; Protein composition; Proteins; Proteomics; Proteomics - methods; Purity; Secretase; Sphingomyelin; Sphingomyelins - analysis; Sphingomyelins - metabolism; Stem cells; Strategy; Technology; Thermal decomposition; Transport; Versatility
• ISSN: 1744-4292; 1744-4292
• DOI: 10.1038/msb.2011.74

We manufactured a novel type of lipid‐coated superparamagnetic nanoparticles that allow for a rapid isolation of plasma membranes (PMs), enabling high‐resolution proteomic, glycomic and lipidomic analyses of the cell surface. We used this technology to characterize the effects of presenilin knockout on the PM composition of mouse embryonic fibroblasts. We found that many proteins are selectively downregulated at the cell surface of presenilin knockout cells concomitant with lowered surface levels of cholesterol and certain sphingomyelin species, indicating defects in specific endosomal transport routes to and/or from the cell surface. Snapshots of N ‐glycoproteomics and cell surface glycan profiling further underscored the power and versatility of this novel methodology. Since PM proteins provide many pathologically relevant biomarkers representing two‐thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications. A methodology for rapid, high‐purity isolation of plasma membranes using superparamagnetic nanoparticles is described. The method is illustrated with high‐resolution proteomic, glycomic and lipidomic analyses of presenilin‐deficient cells. Synopsis A methodology for rapid, high‐purity isolation of plasma membranes using superparamagnetic nanoparticles is described. The method is illustrated with high‐resolution proteomic, glycomic and lipidomic analyses of presenilin‐deficient cells. One of the major goals of this paper was to establish a robust method for plasma membrane (PM) isolation in order to perform a full analysis of their biomolecular composition. Using thermal decomposition, we manufactured superparamagnetic nanoparticles (SPMNPs) that we rendered water soluble and monodisperse by subsequent coupling of NH 2 phospholipids. When incubated with cell monolayers, these cationic phospholipids‐SPMNPs remain predominately localized at the cell surface. We applied these unexpected feature of phospholipids‐SPMNPs to establish a novel protocol to isolate high yields of highly pure PMs. Due to the superb quality and quantity of isolated PM fractions, we could perform a comprehensive and comparative biomolecular profiling on this subcellular compartment that included proteomics, N ‐glycoproteomics, lipidomics and N ‐glycan profiling. This method was subsequently applied to compare the biomolecular composition of PMs isolated from wild‐type and presenilin‐deficient and human prese