Analysis of Entamoeba histolytica Membrane Proteome Using Three Extraction Methods

Abstract

Entamoeba histolytica membrane proteins are important players toward the pathogenesis of amoebiasis, but the roles of most of the proteins are not fully understood. Since efficient protein extraction method is crucial for a successful MS analysis, three extractions methods are evaluated for the use in studying the membrane proteome of E. histolytica: Two commercial kits (ProteoExtract from Calbiochem and ProteoPrep from Sigma), and a conventional laboratory method. The results show that ProteoExtract and the conventional method gave higher protein yields compared to ProteoPrep. LC–ESI–MS/MS identifies 456, 482, and 551 membrane fraction proteins extracted using ProteoExtract, ProteoPrep, and a conventional method, respectively. In silico analysis predicts 108 (21%), 235 (45%), and 177 (34%) membrane proteins from the extracts of ProteoExtract, ProteoPrep, and the conventional method, respectively. Furthermore, analysis of the cytosolic and membrane fractions shows the highest selectivity of the membrane proteins using the ProteoPrep extraction kit. Overall, this study reports 828 E. histolytica membrane fraction proteins that include 249 predicted membrane proteins. The data are available via ProteomeXchange with identifier PXD010171.

Entamoeba histolytica: Proteomics Bioinformatics Reveal Predictive Functions and Protein–Protein Interactions of Differentially Abundant Membrane and Cytosolic Proteins

“…MS data of LFQ from three different extraction methods by Ujang et al, (2018) [ 7 ] were retrieved from ProteomeXchange with identifier no. PXD010171 for differential protein abundant analysis in membrane versus cytosolic fractions using PERSEUS MaxQuant software (MaxPlanck Institute of Biochemistry, Martinsried, Germany).…”

“…Critical steps for protein analysis in a biological context are extraction and isolation of proteins from chemical and physical interactions with other biomolecules from specific cells [ 7 ]. Hence, our previous extensive study (Ujang et al, (2018) [ 7 ]) performed three different extraction methods to fractionate membrane and cytosolic proteins of E. histolytica trophozoites. The extractions were performed to isolate the total membrane and not for an organelle specific-membrane fraction.…”

“…The result showed a commercial kit, ProteoPrep (Sigma-Aldrich, Darmstadt, Germany) was the best method for the membrane protein extraction in terms of its sensitivity and specificity by analyzing membrane and cytosolic fractions. Mixed membrane and cytosolic proteins accounted for 267, 127 and 130 identified proteins in both fractions [ 7 ]. The cytosolic proteins were found to be present in the membrane fraction from the three extraction methods.…”

“…Using the previous MS data of label-free quantification [ 7 ], we aimed to analyze the differential abundance proteins in membrane versus cytosolic fractions of E. histolytica. The differentially abundant proteins were analyzed using bioinformatics platforms to further understand the predictive biological functions and interactions among the proteins.…”

Proteomic Insights into the Biology of the Most Important Foodborne Parasites in Europe

“…Other teams focused on investigating phagosome composition and proteins associated with the phagocytosis process [ 65 ], the cell surface protein profile [ 66 ], and a nuclear and cytoplasmic proteomes of trophozoites [ 67 ]. In a more recent study, the membrane proteome of E. histolytica was described [ 68 ], and the excretory-secretory proteins were identified [ 69 ]. The components of trophozoite ER and Golgi apparatus were characterized as well [ 70 ].…”