Comparative proteomic analysis of extracellular vesicles (EVs) from the hyperthermophilic archaeon, Thermococcusonnurineus NA1

Abstract

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, is able to produce extracellular vesicles (ToEVs) under anaerobic cultivation at 80 oC. EVs were purified through ultra membrane filtration and CsCl-density gradient ultracentrifugation. Two major bands (B & C) were obtained and theirs proteomes were further analyzed. ToEVs were spherical in shape and the size varied from 150 nm to 250 nm in diameter, and the vesicles of both band showed identical shape and size. The proteins of these two kinds of ToEVs were separated and identified by one-dimensional SDS-PAGE and the quantitative proteome analysis of proteins digested by trypsin was performed by LC MS/MS spectrometry. It was revealed that the ToEVs of band B and C contained 294 and 426 proteins, respectively. According to the sub-cellular localization predictions by PSORTb v3, ToEVs proteins consist of cytoplasmic protein, cytoplasmic membrane protein, cell wall protein, extracellular protein and various unknown proteins of the parental cell. Ratio of membrane protein to total protein in bands B and C ToEVs was slightly higher than that of parental cells. The analysis of predicted metabolic function of proteins from two major bands based on the COG (http://www.ncbi.nlm.nih.gov/COG/) database indicated that there was no big discrepancy between them. Proteomic comparison among parental cell, band B and band C ToEVs using Venn diagram showed ove ultracentrifugation. Two major bands (B & C) were obtained and theirs proteomes were further analyzed. ToEVs were spherical in shape and the size varied from 150 nm to 250 nm in diameter, and the vesicles of both band showed identical shape and size. The proteins of these two kinds of ToEVs were separated and identified by one-dimensional SDS-PAGE and the quantitative proteome analysis of proteins digested by trypsin was performed by LC MS/MS spectrometry. It was revealed that the ToEVs of band B and C contained 294 and 426 proteins, respectively. According to the sub-cellular localization predictions by PSORTb v3, ToEVs proteins consist of cytoplasmic protein, cytoplasmic membrane protein, cell wall protein, extracellular protein and various unknown proteins of the parental cell. Ratio of membrane protein to total protein in bands B and C ToEVs was slightly higher than that of parental cells. The analysis of predicted metabolic function of proteins from two major bands based on the COG (http://www.ncbi.nlm.nih.gov/COG/) database indicated that there was no big discrepancy between them. Proteomic comparison among parental cell, band B and band C ToEVs using Venn diagram showed ove