A Simple and Rapid System for Proteomic Analysis of the Archaeon Candidatus Vulcanisaeta moutnovskia

This protocol describes a rapid protein extraction method for the archaeon Candidatus Vulcanisaeta moutnovskia, which can be also implemented for other archaea. The utilization of two different methods for protein extraction constitute the main step of the protocol. Method I involves the extraction with a multi-chaotropic lysis buffer containing a non-denaturing zwitterionic detergent, most efficient for extracting cytosolic proteins. Method II involves a denaturing anionic detergent allowing total disruption of the membranes and capable of extracting both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins. The big advantage of the methods is to use general laboratory chemicals to make powerful extraction buffers, resulting in high quality and quantity of proteins. The methods probably are usable for any other archaea or microbial cells, and takes about 14-22 h. Following extraction and further protein digestion, 1D-nano Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MSMS) analysis with Triple TOF 5600 and Orbitrap technologies were used for protein identification and further quantification.

Sensitivity of Extracellular Vesicles from Human Blood Serum to Various Detergents

Blood exosomes and microvesicles, collectively known as small extracellular vesicles (sEV), are vesicles about 100-150 nm in size. Small EV are involved in various aspects of signaling in the body; in addition, they can serve as markers of various pathologies. For biochemical studies, vesicle solubilization is often required. We tested the ability of various detergents to dissolve membranes of the sEV. Small EV were isolated from the blood serum of healthy volunteers by gel filtration on Sepharose CL-2B and tried to solubilize them using the anionic detergent DOC (sodium deoxycholate), non-ionic detergent Brij 35 (polyoxyethyleneglycol dodecyl ether), zwitterionic detergent CHAPS (3 - [(3-chloramidopropyl) dimethylammonio] -1-propanesulfonate), and cationic detergent CTAB (cetyl trimethylammonium bromide). The concentration of sEV in the solution was determined by dynamic light scattering. We find DOC is the most effective for sEV solubilization.

Heme Dissociation from Myoglobin in the Presence of the Zwitterionic Detergent N,N-Dimethyl-N-Dodecylglycine Betaine: Effects of Ionic Liquids

We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔGdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) in aqueous solution on the ΔGdissociation values. Absorbance experiments show the ILs had minimal effect on ΔGdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies.