Proteome Integral Solubility Alteration: A High-Throughput Proteomics Assay for Target Deconvolution
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01.11.2019 |
Gaetani M.
Sabatier P.
Saei A.
Beusch C.
Yang Z.
Lundström S.
Zubarev R.
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Journal of Proteome Research |
10.1021/acs.jproteome.9b00500 |
0 |
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© 2019 American Chemical Society. Various agents, including drugs as well as nonmolecular stimuli, induce alterations in the physicochemical properties of proteins in cell lysates, living cells, and organisms. These alterations can be probed by applying a stability- and solubility-modifying factor, such as elevated temperature, to a varying degree. As a second dimension of variation, drug concentration or agent intensity/concentration can be used. Compared to standard approaches where curves are fitted to protein solubility data acquired at different temperatures and drug concentrations, Proteome Integral Solubility Alteration (PISA) assay increases the analysis throughput by 1 to 2 orders of magnitude for an unlimited number of factor variation points in such a scheme. The consumption of the compound and biological material decreases in PISA by the same factor. We envision widespread use of the PISA approach in chemical biology and drug development.
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Combination of low-temperature electrosurgical unit and extractive electrospray ionization mass spectrometry for molecular profiling and classification of tissues
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15.08.2019 |
Sukhikh G.
Chagovets V.
Wang X.
Rodionov V.
Kometova V.
Tokareva A.
Kononikhin A.
Starodubtseva N.
Chingin K.
Chen H.
Frankevich V.
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Molecules |
10.3390/molecules24162957 |
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© 2019 by the authors. Real-time molecular navigation of tissue surgeries is an important goal at present. Combination of electrosurgical units and mass spectrometry (MS) to perform accurate molecular visualization of biological tissues has been pursued by many research groups. Determination of molecular tissue composition at a particular location by surgical smoke analysis is now of increasing interest for clinical use. However, molecular analysis of surgical smoke is commonly lacking molecular specificity and is associated with significant carbonization and chemical contamination, which are mainly related to the high temperature of smoke at which many molecules become unstable. Unlike traditional electrosurgical tools, low-temperature electrosurgical units allow tissue dissection without substantial heating. Here, we show that low-temperature electrosurgical units can be used for desorption of molecules from biological tissues without thermal degradation. The use of extractive electrospray ionization technique for the ionization of desorbed molecules allowed us to obtain mass spectra of healthy and pathological tissues with high degree of differentiation. Overall, the data indicate that the described approach has potential for intraoperative use.
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Genetic re-engineering of polyunsaturated phospholipid profile of Saccharomyces cerevisiae identifies a novel role for Cld1 in mitigating the effects of cardiolipin peroxidation
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01.10.2018 |
Lou W.
Ting H.
Reynolds C.
Tyurina Y.
Tyurin V.
Li Y.
Ji J.
Yu W.
Liang Z.
Stoyanovsky D.
Anthonymuthu T.
Frasso M.
Wipf P.
Greenberger J.
Bayır H.
Kagan V.
Greenberg M.
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Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids |
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© 2018 Elsevier B.V. Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL-specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CLOX, has been implicated in the cellular signaling events that initiate apoptosis. CLOX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ12-desaturase gene in yeast, which then synthesized poly unsaturated fatty acids(PUFAs) that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; it also resulted in increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CLOX, and treatment of cells with H2O2 increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.
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Proteomics of mammalian mitochondria in health and malignancy: From protein identification to function
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01.07.2018 |
Eremina L.
Pashintseva N.
Kovalev L.
Kovaleva M.
Shishkin S.
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Analytical Biochemistry |
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3 |
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© 2017 Elsevier Inc. The mitochondrial set of proteins is a dynamic system, crucial for multiple functions of this organelle. Differential expression of genes in various tissues, alternative splicing, post-translational modifications, turnover and spatial dynamics of proteins are the factors that influence mitochondrial proteomes increasing their versatility. A wide range of high-throughput proteomic approaches are extensively used for identification, quantification and functional assessment of human and other mammalian mitochondrial proteins. This article reviews the methods and approaches which can be utilized for achieving one or another specific goal in mitochondrial investigations, and the recent advances in application of proteomics to study the roles of mitochondria in tumorigenesis and cancer progression.
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Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography-Mass Spectrometry
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05.01.2018 |
Mesonzhnik N.
Postnikov P.
Appolonova S.
Krotov G.
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Journal of Proteome Research |
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3 |
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© 2017 American Chemical Society. Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that "spacer peptides" can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-tandem MS methods.
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