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.
2018-03-07 | GSE111488 | GEO
Project description:Transcriptomic analysis of H2S fumigated and Cys treated Arabidopsis seedlings
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:Cysteine (Cys) reversible post-translational modifications (PTMs) are emerging as important players in cellular signaling and redox homeostasis. Here we present Cys-BOOST a novel strategy for LC-MS/MS based quantitative analysis of reversibly modified Cys using switch technique, enrichment via bio-orthogonal cleavable linker and quantification using tandem mas tag (TMT) reagents. We performed direct comparison of Cys-BOOST (n=3) with iodo-TMT (n=3) by analyzing the total CysCys from HeLa cell extracts. As a result higher sensitivity (25,019 vs 9,966 Cys peptides), specificity (98 vs 74 %) and technical reproducibility were obtained by Cys-BOOST. In addition, the application of Cys-BOOST for the analysis of Cys nitrosylation (SNO) in S-nitrosoglutathione (GSNO) treated and non-treated HeLa cell extracts lead to the identification of unprecedented number of SNO proteins (3,537), SNO peptides (9,314) and unique SNO sites (8,304). Based on the quantitative data we describe SNO consensus motifs for endogenous SNO and SNO sites with differential reactivity to GSNO. Collectively, our findings suggest Cys-BOOST as a concurrent method of choice for Cys PTM analysis.
Project description:Thiol-dependent redox regulation is essential for the rapid adaptation of chloroplast metabolism to unpredictable changes of light intensity. The disulfide reductase activity of thioredoxins (Trxs), which relies on photo-reduced ferredoxin (Fdx) and a Fdx-dependent Trx reductase (FTR), constitutes the Fdx-FTR-Trxs system, which links chloroplast redox regulation to light. In addition, chloroplasts harbor an NADPH-dependent Trx reductase (NTR) with a joint Trx domain, NTRC. The activity of these two redox systems is integrated by the balance of the hydrogen peroxide scavenging enzyme 2-Cys peroxiredoxin (2-Cys Prx), which thus plays a key role in maintaining the reducing capacity of chloroplast Trxs in response to light intensity. Based on the severe phenotype of mutant lines lacking NTRC, it is clear that this enzyme plays an essential role in chloroplast redox homeostasis. However, whether the function of NTRC depends on its capacity of reduce 2-Cys Prxs or has additional targets remains unknown. Here, we have addressed this issue by a comparative analysis of the triple mutant of Arabidopsis thaliana, ntrc-2cpab, simultaneously lacking 2-Cys Prxs and NTRC, and the double mutant 2cpab lacking 2-Cys Prxs. The phenotype of the ntrc-2cpab mutant is indistinguishable of the 2cpab mutant, as shown by growth rate, photosynthesis performance, light-dependent redox regulation of enzyme activity and comparative transcriptomics based RNA-Seq. Based on these results, we propose that the function of NTRC in chloroplast redox homeostasis is exerted by the regulation of the redox balance of 2-Cys Prxs rather than by the direct reduction of additional targets.