Computational protocol: Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles

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Protocol publication

[…] Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for label-free proteomic analysis as described []. Briefly, LC-MS/MS (Agilent Technologies Inc., Santa Clara, CA, USA) was used to analyze purified proteins from the (V)LDL and HDL precipitates. The two precipitates (approximately 50 μg each) were resolved in two lanes of a 10% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA), respectively, and subsequently stained with Coomassie blue. Each lane was excised after gel destaining and divided into six pieces equally for in-gel trypsin digestion; after reduction with 20 mm dithiothreitol (DTT) and alkylation using 55 mm iodoacetamide (IAA), each piece was digested with sequencing-grade trypsin (Promega, Madison, WI, USA) overnight. Digested peptides were extracted from the gel, dried, and then desalted. The peptides were analyzed with a Q-Exactive mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled with an Ultimate 3000 RSLC nano-HPLC system (Dionex, Amsterdam, The Netherlands). The raw data were converted to mascot generic (mgf) files using Proteome Discoverer ver. 1.4 (Thermo Scientific, San Jose, CA, USA), and then the mgf files were searched against the Uniprot human proteome database (Released on 29 November 2013; 176,946 sequences, 70,141,034 residues) using an in-house Mascot Server ver. 2.4.1 (Matrix Science, London, UK).Ingenuity Pathway Analysis (IPA®, QIAGEN, Redwood City, CA, USA, Build Version 321501M, Content Version 17199142) was used to determine canonical pathways statistically over-represented in the proteins that were only in the (V)LDL precipitate. Proteins of pathways significantly over-represented were selected for verification based on antibody and recombinant protein availability to set up the multiplex immunoassay. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server, IPA
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Blood Coagulation Disorders, Inherited