Brain Proteome of Drosophila melanogaster Is Enriched with Nuclear Proteins.

The brain proteome of Drosophila melanogaster was characterized by liquid chromatography/high-resolution mass spectrometry and compared to the earlier characterized Drosophila whole-body and head proteomes. Raw data for all the proteomes were processed in a similar manner. Approximately 4000 proteins were identified in the brain proteome that represented, as expected, the subsets of the head and body proteomes. However, after thorough data curation, we reliably identified 24 proteins unique for the brain proteome; 13 of them have never been detected before at the protein level. Fourteen of 24 identified proteins have been annotated as nuclear proteins. Comparison of three used datasets by label-free quantitation showed statistically significant enrichment of the brain proteome with nuclear proteins. Therefore, we recommend the use of isolated brain preparations in the studies of Drosophila nuclear proteins.

Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

The study of the proteome of healthy human blood plasma under conditions of long-term confinement in an isolation chamber.

We identified changes in the proteome of healthy human blood plasma caused by exposure to 105-day confinement in an isolation chamber. After removal of major proteins and concentration of minor proteins, plasma fractions were analyzed by two-dimensional electrophoresis followed by identification of significantly different protein spots by mass spectrometric analysis of the peptide fragments. The levels of α- and ß-chains of fibrinogen, a fragment of complement factor C4, apolipoproteins AI and E, plasminogen factor C1 complement, and immunoglobulin M changed in participants during the isolation period. These changes probably reflect the adaptive response to altered conditions of life.

[Selection of the peptide mass tolerance value for the protein identification with peptide mass fingerprinting].

Peptide mass-fingerprint is widely used for protein identification while studying proteome with the use of 1D or 2D electrophoresis. Peptide mass tolerance indicates the fit of theoretical peptide mass with the experimental measurements, and choice of this parameter sufficiently influences the protein identification. The role of peptide mass tolerance was estimated by counting the number of identified proteins for the reference set of mass-spectra. The reference set of 400 Ultraflex (Bruker Daltonics, Germany) mass-spectra was obtained for the slices of 1D gel of liver microsomes. Using Mascot server for protein identification, the peptide mass tolerance value was varied in the range from 0.02 to 0.40 Da with a step 0.01 Da. Depending on the tolerance the number of identified protein changes up to 10 times. Maximal number of identified proteins was reported for the tolerance value of 0.15 Da (120 ppm), which is 1.5 - 2 times higher than the recommended values for such type of mass-spectrometers. The software program PMFScan was developed to obtain the dependence of number of identified proteins of the tolerance values.

[Study of proteomic profile of Danio rerio embryos using one-dimensional electrophoresis and mass spectrometry].

In the present study, a proteomic technology combining one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry (MALDI-TOF-PMF) has been successfully applied for revelation of changes in the protein profile of zebrafish (Danio rerio) 52 hpf embryos. Prior to 1DE separation of zebrafish embryonic proteins, the procedure for obtaining embryos homogenate was optimized by ultrasonic treatment. A total of 84 proteins, including 15 vitellogenins, were identified. It was shown that growing ofzebrafish embryos in the medium with doxorubicin (DOX) stimulated Caspase-3 induction and promoted the disappearance of cardiac troponins, both these findings being consistent with literature data on doxorubicin-induced cardiotoxicity. The 1DE-based proteomic mapping approach proposed herein enabled not only to identify proteins but also to register those changes in embryos' proteomic profile that were caused by doxorubicin.