Abstract
Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.
Highlights
Two-dimensional gel electrophoresis (2DE) and MALDITOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum
The work presented here gives an overview on the expressed cytosolic proteins and on the macromolecular complexes of T. acidophilum cultured under aerobic growth conditions at 58 °C, pH 1.5–1.8
We used the 2DE-based protein separation and MALDI-TOF MS protein identification method and Coomassie Blue or silver staining to visualize proteins displayed in denaturing gels
Summary
Growth of T. acidophilum and Sample Preparation—T. acidophilum stock (1.5 ml) stored at Ϫ80 °C was inoculated into 50 ml of T. acidophilum medium preheated to 58 °C in an oil bath [7]. After washing twice with MilliQ H2O for 1 min, gels were stained for 20 min in a freshly made solution containing 2 g/liter AgNO3 and 375 l/liter formaldehyde, washed briefly with MilliQ H2O, and developed in a solution including 60 g/liter Na2CO3, 5 mg/liter Na2S2O3, and 250 l/liter formaldehyde until the desired contrast was obtained. This usually took 1–5 min depending on the temperature. Proteins were considered to be identified if the Mascot program returned a Molecular Weight Search (MOWSE) peptide mass database score of at least 44, indicating a probability of less than 5% that the observed match is a random event
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