Despite decades of extensive research, the large-scale analysis of membrane proteins remains a difficult task. This is due to the fact that membrane proteins require a carefully balanced hydrophilic ...and lipophilic environment, which optimum varies with different proteins, while most protein chemistry methods work mainly, if not only, in water-based media. Taking this review Santoni, Molloy and Rabilloud, Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000, 21, 1054-1070 as a pivotal paper, the current paper analyzes how the field of membrane proteomics exacerbated the trend in proteomics, i.e. developing alternate methods to the historical two-dimensional electrophoresis, and thus putting more and more pressure on the mass spectrometry side. However, in the case of membrane proteins, the incentive in doing so is due to the poor solubility of membrane proteins. This review also shows that in some situations, where this solubility problem is less acute, two-dimensional electrophoresis remains a method of choice. Last but not least, this review also critically examines the alternate approaches that have been used for the proteomic analysis of membrane proteins.
Sodium dodecyl sulfate electrophoresis (SDS) is a protein separation technique widely used, for example, prior to immunoblotting. Samples are usually prepared in a buffer containing both high ...concentrations of reducers and high concentrations of SDS. This conjunction renders the samples incompatible with common protein assays. By chelating the SDS, cyclodextrins make the use of simple, dye-based colorimetric assays possible. In this paper, we describe the optimization of the assay, focussing on the cyclodextrin/SDS ratio and the use of commercial assay reagents. The adaptation of the assay to a microplate format and using other detergent-containing conventional extraction buffers is also described.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Toxicoproteomics can be defined as the application of proteomic approaches to the understanding of toxicology problems, and this review deals with the various types of applications that have been ...described in the literature. Toxicoproteomics has been applied to very different classes of toxicants, from drugs and natural products to metals, or from industrial chemicals to nanoparticles and nanofibers. It has also been applied to address questions at different levels, from the search of the primary molecular targets of toxicants to the deciphering of the molecular responses of cells and tissues to toxicants. Although restricted to mammalian cells and tissues, this paper reviews these two levels of investigation and the different application areas of toxicoproteomics, leading to the discussion of the advantages and drawbacks of the most popular proteomic platforms. Some of the pending questions in toxicoproteomics are also critically addressed, such as the specificity, validation, and result hierarchization issues. The question of shared mechanisms, which are encountered in many toxicoproteomic papers dealing with different toxicants, is also discussed. Finally, the future of toxicoproteomics is briefly outlined.
Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap ...equipment and chemicals. It is compatible with downstream processing, such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.
Two-dimensional gel electrophoresis was instrumental in the birth of proteomics in the late 1980s. However, it is now often considered as an outdated technique for proteomics-a thing of the past. ...Although this opinion may be true for some biological questions, e.g., when analysis depth is of critical importance, for many others, two-dimensional gel electrophoresis-based proteomics still has a lot to offer. This is because of its robustness, its ability to separate proteoforms, and its easy interface with many powerful biochemistry techniques (including western blotting). This paper reviews where and why two-dimensional gel electrophoresis-based proteomics can still be profitably used. It emerges that, rather than being a thing of the past, two-dimensional gel electrophoresis-based proteomics is still highly valuable for many studies. Thus, its use cannot be dismissed on simple fashion arguments and, as usual, in science, the tree is to be judged by the fruit.
TiO2 particles are commonly used as dietary supplements and may contain up to 36% of nano-sized particles (TiO2-NPs). Still impact and translocation of NPs through the gut epithelium is poorly ...documented.
We show that, in vivo and ex vivo, agglomerates of TiO2-NPs cross both the regular ileum epithelium and the follicle-associated epithelium (FAE) and alter the paracellular permeability of the ileum and colon epithelia. In vitro, they accumulate in M-cells and mucus-secreting cells, much less in enterocytes. They do not cause overt cytotoxicity or apoptosis. They translocate through a model of FAE only, but induce tight junctions remodeling in the regular ileum epithelium, which is a sign of integrity alteration and suggests paracellular passage of NPs. Finally we prove that TiO2-NPs do not dissolve when sequestered up to 24 h in gut cells.
Taken together these data prove that TiO2-NPs would possibly translocate through both the regular epithelium lining the ileum and through Peyer's patches, would induce epithelium impairment, and would persist in gut cells where they would possibly induce chronic damage.
Due to the physicochemical properties of nanoparticles, the use of nanomaterials increases over time in industrial and medical processes. We herein report the negative impact of nanoparticles, using ...solid growth conditions mimicking a biofilm, on the ability of Bacillus subtilis to fight against a stress. Bacteria have been exposed to sublethal doses of nanoparticles corresponding to conditions that bacteria may meet in their natural biotopes, the upper layer of soil or the gut microbiome. The analysis of the proteomic data obtained by shotgun mass spectrometry have shown that several metabolic pathways are affected in response to nanoparticles, n-ZnO or n-TiO2, or zinc salt: the methyglyoxal and thiol metabolisms, the oxidative stress and the stringent responses. Nanoparticles being embedded in the agar medium, these impacts are the consequence of a physiological adaptation rather than a physical cell injury. Overall, these results show that nanoparticles, by altering bacterial physiology and especially the ability to resist to a stress, may have profound influences on a "good bacteria", Bacillus subtilis, in its natural biotope and moreover, on the global equilibrium of this biotope.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Silver nanoparticles (Ag-NPs) are widely used as biocides, leading to contamination of the environment and possible adverse effects on humans. Recent studies revealed that the cellular response to ...acute exposure to Ag-NPs differs from the response to chronic exposure, although we currently lack systematic studies comparing responses to different dosing regimens. In this study, A549 lung epithelial cells were exposed to 15 nm NM300K or 59 nm PVP-coated Ag-NPs under two different conditions. Under these two conditions, the cells received the same total sub-lethal concentration of Ag-NPs, but the dose was either administered over a 24 hour period (acute exposure) or split over four successive days (repeated exposure). These two types of Ag-NPs were chosen as PVP-coated particles were hypothesized to dissolve more slowly than NM300K particles. EXAFS measurements confirmed this hypothesis, showing more rapid oxidation of Ag
0
-NPs to Ag
I
in cells exposed to NM300K. The intracellular Ag content was higher in cells exposed to NM300K, and higher in cells following acute exposure than cells exposed to repeated doses. Whatever the exposure scenario, Ag
I
bound to thiol-containing intracellular proteins. Both exposure regimens altered cellular metabolism, caused intracellular ROS accumulation and blocked cell cycle progression. DNA damage was only observed following acute exposure, as strand breaks in cells exposed to NM300K and oxidized DNA bases in cells exposed to Ag-PVP. This damage was concomitant with decreased DNA repair activities. Together, these results show that acute exposure of A549 cells to Ag-NPs induces stronger effects on DNA integrity than repeated exposure. Nevertheless, repeated exposure to a low concentration of Ag-NPs profoundly altered the cell's metabolism and blocked cell cycle progression, confirming that both exposure regimens have detrimental effects.
Acute exposure of A549 cells to Ag-NPs induces stronger effects on DNA integrity, ROS level, cell metabolism and cell cycle than repeated exposure. Ag-NPs dissolves in both exposure conditions and Ag ions recombine with thiolated proteins.
In this second decade of the 21st century, we are lucky enough to have different types of proteomic analyses at our disposal. Furthermore, other functional omics such as transcriptomics have also ...undergone major developments, resulting in mature tools. However, choice equals questions, and the major question is how each proteomic strategy is fit for which purpose. The aim of this opinion paper is to reposition the various proteomic strategies in the frame of what is known in terms of biological regulations in order to shed light on the power, limitations, and paths for improvement for the different proteomic setups. This should help biologists to select the best-suited proteomic strategy for their purposes in order not to be driven by raw availability or fashion arguments but rather by the best fitness for purpose. In particular, knowing the limitations of the different proteomic strategies helps in interpreting the results correctly and in devising the validation experiments that should be made downstream of the proteomic analyses.
The separation of membrane proteins by high-resolution two-dimensional electrophoresis was carried out. At high loads, these proteins are prone to precipitation, resulting in poor resolution. It is ...shown here that the use of thiourea, previously described for focusing in immobilized pH gradients, can be extended to conventional isoelectric focusing. As thiourea inhibits acrylamide polymerization, a modified photopolymerization system must be used. These modifications result in higher solubility of proteins during IEF, thereby increasing the resolution and capacity of the two-dimensional gels.
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