Abstract
Entamoeba histolytica is the protozoan parasite agent of amebiasis, an infectious disease of the human intestine and liver. This parasite contact and kills human cells by an active process involving pathogenic factors. Cellular traffic and secretion activities are poorly characterized in E. histolytica. In this work, we took advantage of a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. A total of 5683 peptides matching with 1531 proteins (FDR of 1%) were identified which corresponds to roughly 20% of the total amebic proteome. Bioinformatics investigations searching for domain homologies (Smart and InterProScan programs) and functional descriptions (KEGG and GO terms) allowed this data to be organized into distinct categories. This data represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established. The data are related to [1].
Highlights
Entamoeba histolytica is the protozoan parasite agent of amebiasis, an infectious disease of the human intestine and liver
Proteins from the internal acquired membrane fraction of E. histolytica trophozoites were treated to obtain tryptic peptides. These were separated by HPLC coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific)
The HPLC was coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific)
Summary
Proteins from the internal membrane fraction (50 mg) were precipitated with the methanol–. Sample were diluted with 25 mM NH4HCO3 to a final concentration of 1 M urea and digested overnight at 37 1C with sequencing grade trypsin gold (1 mg, Promega USA). Peptide mixtures were acidified to pH 2.8 with formic acid and desalted with minispin C18 columns (Nestgrp, USA). The tryptic peptide samples (1 ml roughly containing 1 mg) were separated by reverse-phase chromatography for each experiment via Thermo Scientific Proxeon nano LC using a C18 picofrit analytical column (360 μm OD, 75 μm ID, 10 μm tip, Magic C18 resin, 5 mm size, Newobjective, USA). The HPLC was coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). The mass spectra were acquired in the LTQ Orbitrap velos with full MS scan (RP 30,000) followed by 10 data-dependent MS/MS scans with detection of the fragment ions in the FTMS HCD mode (RP 7500).
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