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

Abstract

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.

Genetic predisposition for beta cell fragility underlies type 1 and type 2 diabetes

“…The generated peptides were labeled in 20 mM of the different NHS esters. Equal amounts of peptide material were mixed, and peptides were fractionated by reversed-phase HPLC 66,67 . Then, peptide fractions eluted 20 min apart were pooled to reduce the number of LC-MS/MS runs to 20 per sample.…”

Brain Lipids in the Pathophysiology and Treatment of Alzheimer’s Disease

“…Furthermore, individuals carrying the ApoE4 allele accumulate less ApoE lipoprotein in the brain than non-ApoE4 carriers [38]. Hence, the expression of ApoE4 in SAD cases appears to alter cholesterol homeostasis in neurons in a similar way as that induced by γ-secretase loss-of-function in PS1-deficient cells and transgenic models of AD harboring clinical PS1 mutations [33,34]. In such AD models, the loss of γ-secretase activity leads to impaired uptake of lipoproteins from the extracellular media due to the poor internalization of ApoE receptors like the LDLR (low-density lipoprotein receptor) [34].…”

“…On the other hand, cholesterol and SM storage disorders impair intracellular trafficking of APP, resulting in the accumulation of APP, APP-CTFs, and Aβ in autophagic vesicles of the endolysosomal pathway [54,55]. Accordingly, impaired distribution of cholesterol and SM is accompanied by the downregulation of proteins involved in endosomal redistribution and fusion to the plasma membrane (SNAREs and RABs) in PS1-deficient cells [33]. These evidences suggest that dysfunctional vesicular trafficking between the plasma membrane and intracellular compartments may be caused by membrane lipid alterations that lead to the neuritic pathology and altered APP processing in FAD transgenic models [33,56].…”

“…These mutations may exert additional effects on cellular signaling as a consequence of the altered processing of certain membrane proteins that could influence lipid cellular homeostasis. Interestingly, γ-secretase loss of function induced by the ablation of PSs or by transgenic expression of PS1 mutants provoked a severe imbalance in the cholesterol content of the plasma membrane and intracellular membranes [33,34]. In this sense, PS ablation increased the overall levels of cholesterol and sphingomyelin (SM) in cells, whereas the local concentration of cholesterol at the plasma membrane was dramatically reduced, resulting in the intracellular accumulation of cholesterol and cholesterol-rich membrane domains, such as lipid rafts [33,34].…”

“…Accordingly, impaired distribution of cholesterol and SM is accompanied by the downregulation of proteins involved in endosomal redistribution and fusion to the plasma membrane (SNAREs and RABs) in PS1-deficient cells [33]. These evidences suggest that dysfunctional vesicular trafficking between the plasma membrane and intracellular compartments may be caused by membrane lipid alterations that lead to the neuritic pathology and altered APP processing in FAD transgenic models [33,56]. Additional studies have also linked shingolipid lysosomal accumulation to autophagic dysfunction and dystrophic neurite formation in AD [55,57].…”