Peptide-mediated ‘miniprep’ isolation of extracellular vesicles is suitable for high-throughput proteomics

Publication date: Available online 22 February 2016 Source:EuPA Open Proteomics Author(s): Jaco C. Knol, Inge de Reus, Tim Schelfhorst, Robin Beekhof, Meike de Wit, Sander R. Piersma, Thang V. Pham, Egbert F. Smit, Henk M.W. Verheul, Connie R. Jiménez Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since EVs are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, they represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation are laborious, time-consuming, and require high inputs. Ghosh and co-workers recently described an isolation method utilizing Heat Shock Protein (HSP)-binding peptide Vn96 to aggregate HSP-decorated EVs, which can be performed at small ‘miniprep’ scale. Based on microscopic, immunoblot, and RNA sequencing analyses this method compared well with ultracentrifugation-mediated EV isolation, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cell-conditioned medium. Despite a 30-fold different scale (ultracentrifugation: 60ml/Vn96-mediated aggregation: 2ml) both methods yielded comparable numbers of identified proteins (3115/3085), with similar reproducibility of identification (72.5%/75.5%) and spectral count-based quantification (average ...

Source: EuPA Open Proteomics - February 23, 2016 Category: Bioinformatics Source Type: research