Project description:The absolute protein amounts for the selected yeast ribosomal proteins (RPs) were determined using the standard peptides and TMT labelling. The range of the standard tryptic peptides were selected and synthesized based on the preliminary data-dependent experiments. The synthetic peptides were mixed and four aliquots containing different amounts of the peptides were labelled each with a different TMT labelling reagent. The yeast cell lysate was digested and labelled in parallel. The labelled cellular and standard samples were combined, and the absolute peptide amounts in the lysate were calculated by comparison to the known amounts in the standard TMT channels.
Project description:Hela cells were treated with a mixture of amino acids and other small molecules, called active mixture for various time periods. The aim was to identify the differentially expressed genes that are upregulated or downregulated upon treatment at different time points.
Project description:Protein extracts of Saccharomyces cerevisiae CEN.PK113-7D cultivated in chemostats under different conditions. Representative samples containing aliquots of all conditions were spiked with UPS2 standard (Sigma) to estimate absolute values in fmol. The conditions for Saccharomyces cerevisiae CEN.PK113-7D are: T2- Standard condition : 30°C, pH 5.5 T3- High temperature: 36°C, pH 5.5 T4- Low pH: 30°C, pH 3.5 T5- Osmotic stress : 30°C, pH 5.5, 1M KCl T6- Anaerobic condition Furthermore, representative samples pooling aliquots of each condition are indicated as "bulk" samples. These samples were spiked with UPS proteins. A validation step was carried out by spiking 4 external proteins at known concentrations within the yeast and UPS proteins mixture
Project description:Monitoring of host cell proteins (HCPs) during the manufacture of monoclonal antibodies (mAb) has become a critical requirement to provide effective and safe drug product. ELISA assays are still the current gold standard for the quantification of protein impurities. However, this technique has several limitations and does, among others, not enable the precise identification of proteins. In this context, mass spectrometry (MS) has emerged as an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs. However, in order to be routinely implemented in biopharmaceutical companies, liquid chromatography (LC)-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification. In this study, we developed a promising MS-based analytical workflow coupling the use of an innovative quantification standard, the HCP Profiler solution, with a spectral library-based data-independent acquisition (DIA) method and strict data validation criteria. The performance of the HCP Profiler solution was compared to more conventional standard protein spikes and the DIA approach was benchmarked against classical data-dependent acquisition (DDA) using samples collected at various stages of the manufacturing process. Finally, we further explored the possibility to use spectral library-free DIA. As a result, our method showed accurate and reproducible (coefficients of variation (CVs) < 10%) quantification of HCPs while reaching a sensitivity down to the sub-ng/mg mAb level.
Project description:To see DNA methylation-repressed genes, we did RNA-seq in 5-aza treated cells and control cells. And we also did P65 ChIP-seq to see P65-dependent expressions.