An Integrated Approach to Explore Composition and Dynamics of Cholesterol-rich Membrane Microdomains in Sexual Stages of Malaria Parasite

Abstract

Membrane microdomains that include lipid rafts, are involved in key physiological and pathological processes and participate in the entry of endocellular pathogens. These assemblies, enriched in cholesterol and sphingolipids, form highly dynamic, liquid-ordered phases that can be separated from the bulk membranes thanks to their resistance to solubilization by nonionic detergents. To characterize complexity and dynamics of detergent-resistant membranes of sexual stages of the rodent malaria parasite , here we propose an integrated study of raft components based on proteomics, lipid analysis and bioinformatics. This analysis revealed unexpected heterogeneity and unexplored pathways associated with these specialized assemblies. Protein-protein relationships and protein-lipid co-occurrence were described through multi-component networks. The proposed approach can be widely applied to virtually every cell type in different contexts and perturbations, under physiological and/or pathological conditions.

Joint action of miR‐126 and MAPK/PI3K inhibitors against metastatic melanoma

“…Proteins were identified with a minimum of one peptide rank = 1. Protein abundance was determined according to the label‐free Top3 method as already described (Fratini et al ., ).…”

Proteomic analysis of plasma exosomes from Cystic Echinococcosis patients provides in vivo support for distinct immune response profiles in active vs inactive infection and suggests potential biomarkers

“…Fractions in the density range 1.07–1.20 ± 0.02 g/ml were analysed by label free quantitative (LFQ) mass spectrometry. The Protein Abundance Profiles (PAPs) of active CE pools (AP), inactive CE pools (IP) and control pools (Ctr) were calculated as described [ 33 ]. Proteins identified in at least two replicas and reporting PAP Pearson correlation values R ≥ 0.7 (p ≤ 0.05) were selected among AP and IP (APs, IPs).…”

“…Liquid Chromatography Tandem Mass Spectrometry. Protein identification and Protein Abundance Profiles (PAPs) were performed as already described [33] with some improvements. Briefly, 1/10 (5μl) of each sample was used for silver-staining on 4-12% Nu-PAGE gels (Invitrogen; Thermo Fisher Scientific, Inc.) to perform a quality control evaluating the protein mixture complexity and normalization (S4 Fig in S1 File); the remaining amount was resolved on 12% SDS-PAGE for 4 cm and the Coomassie stained lanes were divided into 8 bands.…”

“…Proteins identified in AP and/or IP were subjected to a stringent filtering step based on protein abundance profile (PAP, [33]) correlation clustering (S6 Fig in S1 File and S2 Table). Thus, for AP and IP, only those proteins that showed abundance profiles (across the density fractions) that were consistent (R � 0.7) in at least two replicas were selected for further analysis.…”

Caveolin-1 Endows Order in Cholesterol-Rich Detergent Resistant Membranes

“…In-gel tryptic digestion was performed as already described [26] with slight modifications. Briefly, gel slices were destained in a mixture 1:1 of acetonitrile (ACN)/50 mM ammonium hydrogen carbonate (AHC); protein cysteines were reduced with 10 mM DTT/25 mM ACH (30 min at 56 °C) and alkylated by 55 mM Iodoacetamide/25 mM ACH (30 min in the dark at RT).…”