Computational protocol: Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex specific differences in cargo and a sensitivity to ivermectin

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Protocol publication

[…] Proteomic analysis of EV preparations was performed commercially (System Biosciences, Palo Alto, CA). Briefly, purified EV preparations were resuspended in RIPA Buffer (2.0% SDS, 150 mM NaCl, 50 mM Tris, pH 8.5, 1X Roche cOmplete Protease Inhibitor Cocktail [Sigma-Aldrich]) and heated at 100°C for 15 minutes, clarified by centrifugation and the protein concentration determined by Qubit fluorometry (Invitrogen). 8–10 μg of protein was processed by SDS-PAGE using 10% Bis-Tris NuPage mini-gel (Invitrogen). Following in-gel tryptic digestion at 37°C for 4 h, samples were analyzed by nano LC-MS/MS with a Waters NanoAcquity HPLC System (Waters Corp. Milford, MA) interfaced to a ThermoFisher Q Exactive. Spectral data were searched against copies of the UniProt B. malayi, Ascaris suum and Caenorhabditis elegans databases (UniProt release 2017_4) using a locally running copy of MASCOT v2.6 (Matrix Science Ltd., London, UK). Common mammalian contaminants were also included in this step and positive matches removed from downstream analysis. The search was restricted using the following parameters; maximum missed cleavages = 2, fixed modifications = carbamidomethyl (C), variable modifications = Oxidation (M), Acetyl (N-term), Pyro-Glu (N-term Q) and Deamidation (N, Q), a peptide mass tolerance of 10 ppm, and a fragment mass tolerance of 0.02 Da. Mascot DAT files were parsed into the Scaffold software for validation, and filtered to create a non-redundant list from which mammalian contaminants were subtracted. Data were filtered using 1% protein and peptide FDR and requiring at least two unique peptides per protein. Protein sets were compared using UpSetR (40), a novel web-based technique to analyze discrete data sets and their intersections. GO analysis was performed using Blast2GO v.4 (BioBam Bioinformatics, Valencia, Spain), a program that extracts GO terms from remote BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of input queries and from InterPro motifs identified via InterProScan (www.ebi.ac.uk) to generated a list of validated GO annotations. The transmembrane topology of identified proteins was predicted using locally installed HMMTOP v.2.1. […]

Pipeline specifications

Software tools UpSet, Blast2GO, InterPro, InterProScan, HMMTOP
Organisms Caenorhabditis elegans, Brugia malayi, Homo sapiens, Mus musculus, Canis lupus familiaris, Dirofilaria immitis
Diseases Elephantiasis, Filarial, Multiple Sclerosis, Nematode Infections, Discrete Subaortic Stenosis
Chemicals Ivermectin