On the utility of ultrafast MS1-only proteomics in drug target discovery studies based on thermal proteome profiling method.

Advances in high-throughput high-resolution mass spectrometry and the development of thermal proteome profiling approach (TPP) have made it possible to accelerate a drug target search. Since its introduction in 2014, TPP quickly became a method of choice in chemical proteomics for identifying drug-to-protein interactions on a proteome-wide scale and mapping the pathways of these interactions, thus further elucidating the unknown mechanisms of action of a drug under study. However, the current TPP implementations based on tandem mass spectrometry (MS/MS), associated with employing lengthy peptide separation protocols and expensive labeling techniques for sample multiplexing, limit the scaling of this approach for the ever growing variety of drug-to-proteomes. A variety of ultrafast proteomics methods have been developed in the last couple of years. Among them, DirectMS1 provides MS/MS-free quantitative proteome-wide analysis in 5-min time scale, thus opening the way for sample-hungry applications, such as TPP. In this work, we demonstrate the first implementation of the TPP approach using the ultrafast proteome-wide analysis based on DirectMS1. Using a drug topotecan, which is a known topoisomerase I (TOP1) inhibitor, the feasibility of the method for identifying drug targets at the whole proteome level was demonstrated for an ovarian cancer cell line.

Ultra-Fast Mass Spectrometry in Plant Biochemistry: Response of Winter Wheat Proteomics to Pre-Sowing Treatment with Iron Compounds.

In recent years, ultrafast liquid chromatography/mass spectrometry methods have been extensively developed for the use in proteome profiling in biochemical studies. These methods are intended for express monitoring of cell response to biotic stimuli and elucidation of correlation of molecular changes with biological processes and phenotypical changes. New technologies, including the use of nanomaterials, are actively introduced to increase agricultural production. However, this requires complex approbation of new fertilizers and investigation of mechanisms underlying the biotic effects on the germination, growth, and development of plants. The aim of this work was to adapt the method of ultrafast chromatography/mass spectrometry for rapid quantitative profiling of molecular changes in 7-day-old wheat seedlings in response to pre-sowing seed treatment with iron compounds. The used method allows to analyze up to 200 samples per day; its practical value lies in the possibility of express proteomic diagnostics of the biotic action of new treatments, including those intended for agricultural needs. Changes in the regulation of photosynthesis, biosynthesis of chlorophyll and porphyrin- and tetrapyrrole-containing compounds, glycolysis (in shoot tissues), and polysaccharide metabolism (in root tissues) were shown after seed treatment with suspensions containing film-forming polymers (PEG 400, Na-CMC, Na2-EDTA), iron (II, III) nanoparticles, or iron (II) sulfate. Observations at the protein levels were consistent with the results of morphometry, superoxide dismutase activity assay, and microelement analysis of 3-day-old germinated seeds and shoots and roots of 7-day-old seedlings. A characteristic molecular signature involving proteins participating in the regulation of photosynthesis and glycolytic process was suggested as a potential marker of the biotic effects of seed treatment with iron compounds, which will be confirmed in further studies.

Yeast Ribonucleotide Reductase Is a Direct Target of the Proteasome and Provides Hyper Resistance to the Carcinogen 4-NQO.

Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.

Proteomics-based scoring of cellular response to stimuli for improved characterization of signaling pathway activity.

Omics technologies focus on uncovering the complex nature of molecular mechanisms in cells and organisms, including biomarkers and drug targets discovery. Aiming at these tasks, we see that information extracted from omics data is still underused. In particular, characteristics of differentially regulated molecules can be combined in a single score to quantify the signaling pathway activity. Such a metric can be useful for comprehensive data interpretation to follow: (1) developing molecular responses in time; (2) potency of a drug on a certain cell culture; (3) ranking the signaling pathway activity in stimulated cells; and (4) integration of the omics data and assay-based measurements of cell viability, cytotoxicity, and proliferation. With recent advances in ultrafast mass spectrometry for quantitative proteomics allowing data collection in a few minutes, proteomics score for cellular response to stimuli can become a fast, accurate, and informative complement to bioassays. Here, we utilized an interquartile-based selection of differentially regulated features and a variety of schemes for quantifying cellular responses to come up with the quantitative metric for total cellular response and pathway activity. Validation was performed using antiproliferative and virus assays and label-free proteomics data collected for cancer cells subjected to drug stimulation.