Project description:Recent advances in mass spectrometry (MS) have enabled quantitative proteomics to become a powerful tool in the field of drug discovery, especially when applied toward proteome-wide target engagement studies. Similar to temperature gradients, increasing concentrations of organic solvents stimulate unfolding and precipitation of the cellular proteome. This property can be influenced by physical association with ligands and other molecules, making individual proteins more or less susceptible to solvent-induced denaturation. Herein, we report the development of proteome-wide solvent shift assays by combining the principles of solvent-induced precipitation (Zhang et al., 2020) with modern quantitative proteomics. Using this approach, we developed solvent proteome profiling (SPP), which is capable of establishing target engagement through analysis of SPP denaturation curves. We readily identified the specific targets of compounds with known mechanisms of action. As a further efficiency boost, we applied the concept of area-under-the-curve analysis to develop solvent proteome integral solubility alteration (solvent-PISA) and demonstrate that this approach can serve as a reliable surrogate for SPP. We propose that by combining SPP with alternative methods, like thermal proteome profiling, it will be possible to increase the absolute number of high-quality melting curves that are attainable by either approach individually thereby increasing the fraction of the proteome that can be screened for evidence of ligand binding.
Project description:Effect of DNA extraction methods on the determination of the structure of microbial communities in the phosphogypsum waste heap soil
Project description:Entamoeba histolytica membrane proteins are important players in the parasite’s pathogenicity. However, most of the proteins have not been identified. This study reports the membrane proteins extracted using three extractions methods: two commercial kits (ProteoExtract® from Calbiochem and ProteoPrep® from Sigma), and a conventional laboratory method. The resulting membrane fractions (MF) and cytosolic fractions (CF)were analysed using LC-ESI-MS/MS. The proteins identified in at least two out of three biological replicates revealed a total of 490, 492, and 587 MF proteins extracted using the ProteoExtract® kit, ProteoPrep® kit and conventional method, respectively. Meanwhile, 487, 611 and 343 proteins were identified in the CF extracted using the ProteoExtract® kit, ProteoPrep® kit and conventional method, respectively. Analysis of the identified MF and CF proteins extracted by the respective extraction kits suggests that the ProteoPrep® extraction kit was the most selective in separating MF and CF among the three extraction methods.
Project description:Human stool samples were collected, microbial cells were harvested and subjected to protein extraction using different protocols for metaproteomics analyses. The protein extraction methods differ from the usage of different lysis buffer (SDS, urea and commercially available B-Per reagent), bead beating, ultrasonication and heating. The impacts of different protein extraction methods on metaproteomics results were evaluated with multiple criteria including protein yields, peptide identifications, taxonomic compositions and functional compositions.