Project description:Identification of targets of the protein disulfide reductase thioredoxin using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and thiol specific differential labeling with isotope-coded affinity tags (ICAT). Reduction of specific target disulfides is quantified by measuring ratios of cysteine residues labeled with the heavy (13C) and light (12C) ICAT reagents in peptides derived from tryptic digests of Trx-treated and non-treated samples. Keywords: protein, LC-MS/MS, ICAT
Project description:The transcription level of a rex-deficient S. aureus mutant in comparison to its parental strain S. aureus SH1000 was analyzed using DNA microarrays. S. aureus N315 microarrays were purchased from Scienion (Scienion AG, Berlin, Germany) and were produced by spotting 2,338 PCR products of the 2,593 ORFs comprising annotated genome of S. aureus N315 [reference identification: NC_002745] on a glass slide. Each ORF is present in duplicate on the microarray (further details can be found at http://www.scienion.com), cDNA was synthesized from total RNA with the LabelStar Array Kit from QIAGEN using the QIAGEN protocol with slight modifications: Random hexamer primer were used (Invitrogen, Karlsruhe, Germany) and Cy3- and Cy5-dCTP were purchased from Perkin-Elmer (Rodgau - Juegesheim, Germany). As recommended by Scienion, 10 µg RNA from either SH1000 or AK1 were used for cDNA synthesis. After hybridisation for 72 h, the microarrays were washed as recommended by the manufacturer. Data analysis. The hybridized microarrays were scanned with a GenePix 4000B microarray scanner (MDS Analytical Technologies GmbH, Ismaning, Germany). A geometric raster was laid over the resulting microarray picture to distinguish the signals from the background. After localization of single spots, intensities and global background were calculated automatically. The hybridization patterns and intensities were quantitatively analyzed using the Imagene 6 software (BioDiscovery, El Segundo, CA). The replicates were averaged, and the spots identified by Imagene 6 (BioDiscovery) as flawed were omitted. The data set was normalized by application of the LOWESS algorithm. In a next step, the intensity values of all arrays for each time point as well as for all time points combined were used for t tests. Genes with a change of <0.5- or â?¥2.0-fold were characterized as having significantly differing amounts of transcripts based on t tests with a P value cut-off of at least 0.05. Gene functions were assigned to the respective accession numbers and annotations as compiled on DOGAN, a web page for S. aureus N315 (http://www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=n315G1). The parental strain SH1000 and the Rex deficient mutant AK1 were applied on full-genome microarrays to get a detailed view on the differences in the transcriptional profiles which are caused â?? directly or indirectly â?? by the introduced mutation. More specifically, expression levels were compared at five time points, covering different growth phases. To highlight the general changes in the expression profile between SH1000 and the rex mutant, the microarray data of all five time points were also analyzed in a combined way using standard statistical methods. In further experiments, we focused on those genes, which seemed to flag the general difference between the investigated strains.
Project description:To identify the target of the monoclonal antibody, Immuoprecipitation (IP) was performed to pull-down target protein from cell culture extraction, corn or zebrafish tissue lysate. The IP product was validated on silver-stained SDS-PAGE and Western blot. The target band was cut out and performed LC-MS/MS for target protein identification.
Project description:Constitutional MLH1 epimutations are a rare cause of Lynch syndrome. Low methylation levels (≤10%) have been occasionally described. The aim of this study was the identification of patients with low levels of epigenetic mosaicism in MLH1 gene. Eighteen patients with MLH1 hypermethylated tumors and undetectable methylation in blood as assessed by Methylation-Specific Multiplex Ligation-Dependent Probe Amplification were included (MS-MLPA). Highly sensitive MS-Melting Curve Analysis (MS-MCA) at MLH1 promoter was used to screen for epigenetic mosaicism. Constitutional methylation was confirmed by other methods. Mutational analysis of hereditary cancer genes including MLH1 was performed. MS-MCA analysis identified one case (5.6%) with low levels of methylation (1-2%) in blood DNA. The patient had developed 3 gastrointestinal tumors at ages 22, 24 and 25, sharing MLH1 promoter hypermethylation and loss of heterozygosity associated with c.655A allele. The presence of low MLH1 methylation levels was confirmed by clonal bisulfite sequencing, evidencing the association with c.-93G allele (in phase with c.655G). The extension of the hypermethylated region overlaps with the reported in constitutional MLH1 epimutation carriers. No rare germline variants were identified. The use of highly sensitive techniques such as MS-MCA has demonstrated to be useful for the detection of low MLH1 methylation levels in blood.
Project description:To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over‑expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper‑mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. Our proteome and transcriptome analyses have been performed during stationary‑phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets. Keywords: Molecular markers, antibiotic resistance, glycopeptides, growth-phase
Project description:CHD (chromodomain helicase DNA binding protein) family is composed of nine members of chromatin remodeling factors that regulate chromatin structure in an ATP-dependent manner. Among them, CHD4 contributes to many basic cellular functions during development mainly through multiple proteins interacting with CHD4 including NuRD (nucleosome remodeling and deacetylase activities) complex, which contains histone deacetylase HDAC1/2. However, functions of CHD4 that are not mediated by NuRD complexes have also been found, implying the existence of unknown proteins that are associated with CHD4. In the present study, we generated CHD4 FLAG-tag knock-in cells in HeLa-S3 and HEK293T cells and sought proteins bound to CHD4 with the use of immunoprecipitation and liquid chromatography and tandem MS (LC-MS/MS) analysis. We found that LCORL (ligand dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like) were reproducibly identified as a novel CHD4 interactors. RNA-sequencing analysis of HEK293T cells depleted of CHD4, LCORL, and NOL4L revealed consistent upregulation of genes related to the Notch pathway. Our results thus suggest that both NOL4L and LCORL may cooperate with CHD4 to suppress the Notch pathway in mammalian cells.
Project description:We applied 4-thiouridine to cultured cells expressing the FLAG/HA-tagged RNA-binding proteins (RBPs) followed by UV 365 nm irradiation. The crosslinked RNA-protein complexes were isolated by immunoprecipitation, and the covalently bound RNA was partially digested with RNase T1 and radiolabeled. The radiolabeled RNPs were subsequenctly separated by SDS-PAGE, the crosslinked RNA segments recovered and converted into a cDNA library and sequenced.
Project description:Gene and protein expression in the breast varies relative to menopausal status (MS) and menstrual phase (MP), but cancer risk biomarker research using archived benign breast samples is usually uninformed by MS-MP data. We hypothesized that gene expression variation relative to ambient hormones can be exploited to develop markers of MS-MP that can be measured directly in breast tissue. Random fine needle aspiration samples from the unaffected breasts of 18 women were utilized for Illumina microarray analysis. Gene expression was correlated with simultaneous serum levels of estradiol, progesterone, and follicle stimulating hormone (FSH). True MS-MP states were defined by age and serum hormones. Genes highly correlated to serum hormones were used to classify samples into 2 MS groups: premenopause or postmenopause. The premenopausal samples were further classified into 3 MP groups: early follicular, late follicular or luteal.