Project description:The objective of the present investigation was to consider the level of variation in the protein expression patterns of closely related Salmonella serovars, in order to search for protein factors with levels of expression or posttranslational modifications characteristic for each serovar. For the comparative expression analysis we have utilised classic 2D GE approach which revealed several proteins with serovar specific expression as well as proteins which do not alter their expression levels between serovars and strains. The proteins of interest were identified using LC/MS/MS. Keywords: 2D GE, MS/MS Analysis of 12 strains of S. enterica representing five different serovars.
Project description:786-O WT and BAP1-KO cells were cultured in heavy (13C6-L-arginine, 13C6-L-lysine, Cambridge Isotope Lab) and light (unlabeled L-arginine and L-lysine) SILAC RPMI-1640 medium (Thermo Fisher Scienific), respectively. After more than 10 passages, cells were lysed with 8 M urea and 5 mg protein from each group were mix. The ubiquitinated peptide enrichment was performed using PTMScan® (Cell Signaling Technology, #5562) following the manufacturer’s instructions.
Project description:October 2013 surface seawater collected from Monterey Bay was incubated with 1 micromolar 13C labeled glucose, starch, acetate, lipids, protein, or amino acids for 12 hours. Community RNA was extracted and hybridized to a Roche Nimblegen microarray and analyzed by NanoSIMS to obtain isotope ratio data for all probe spots. Two Chips for fluorescence, and 15 Chips for different substrates from samples incubated for 12 or 36 hours.
Project description:In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and we present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. Here we show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.
Project description:Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Recent studies have demonstrated strong genetic associations between AMD and single nucleotide polymorphisms (SNPs) within genes such as CFH and HTRA1. However, we found monozygotic twins had discordant AMD phenotypes (one with disease, the other without disease), suggesting that an epigenetic mechanism may control the pathogenesis of AMD. We obtained genomic DNA from the twins' peripheral blood mononuclear cells (PBMCs) and subjected it to DNA methylation-chip analysis (MeDIP-chip) that profiled genome-wide DNA methylation patterns on promoters of all genes and microRNAs. Our MeDIP-chip analysis identified 256 genes with hypo-methylated promoters only in the twins with AMD and 744 genes with hyper-methylated promoters only in the twins with AMD. Importantly, the promoter region of IL17RC was associated with hypo-methylated CpG sites only in the twins with AMD but not in the twins without AMD. Two pairs of twins with discordant AMD phenotypes. MeDIP-chip analysis of DNA methylation patterns in PBMCs.