Project description:Investigation of whole genome expression pattern of 60 and 72 hours post fertilization Danio Rerio embryos exposed to TMT and vehicle control Embryos were exposed to 10uM TMT or control from 48hpf to 60 or 72 hpf. Three replicates were collected for each time point. 40 embryos were pooled to comprise a replicate.
Project description:In order to investigate the chicken SLCO1B3 gene functin on the liver metabolism, we used the Yimeng blue eggshell and brown eggshell chickens as the chicken liver SLCO1B3 gene knock-down animal to do the proteomic analysis.
Project description:Investigation of whole genome expression pattern of 60 and 72 hours post fertilization Danio Rerio embryos exposed to TMT and vehicle control
Project description:BackgroundAndrographolide (Andro) is a diterpenoid derived from Andrographis paniculate, which has anti-inflammatory, antibacterial, antiviral and hepatoprotective activities. Gram-negative bacterial infections can cause varying degrees of liver injury in chickens, although Andro has been shown to have a protective effect on the liver, its underlying mechanism of action and effects on liver proteins are not known.MethodsThe toxicity of Andro on the viability of leghorn male hepatoma (LMH) cells at different concentrations and times was analyzed by CCK-8 assays. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the culture supernatants were measured using an automatic biochemical analyzer to evaluate the protective effect of androscopolide on LPS-induced injury of LMH cells. Subsequently, TMT proteomics analysis were performed on the negative control group (NC group), LPS, and LPS-Andro groups, and bioinformatics analysis was performed on the differentially expressed proteins (DEPs).ResultsIt was found that Andro reduced ALT and AST levels in the cell supernatant and alleviated LPS-induced injury in LMH cells. Proteomic analysis identified 50 and 166 differentially expressed proteins in the LPS vs. NC group and LPS-Andro vs. LPS group, respectively. Andro may be involved in steroid metabolic processes, negative regulation of MAPK cascade, oxidative stress, and other processes to protect against LPS-induced liver injury.ConclusionsAndro protects against LPS-induced liver injury, HMGCS1, HMGCR, FDPS, PBK, CAV1, PRDX1, PRDX4, and PRDX6, which were identified by differential proteomics, may be the targets of Andro. Our study may provide new theoretical support for Andro protection against liver injury.
Project description:Label free quantification (LFQ) and isobaric labelling quantification (ILQ) are among the most popular protein quantification workflows in discovery proteomics. Here, we compared the TMT 10-plex workflow to label free single shot data-independent acquisition (DIA) method on a controlled sample set. The sample set consisted of ten samples derived from 10 different mouse cerebelli spiked with the UPS2 protein standard in five different concentrations. To match instrument time between the methods, the combined TMT sample was fractionated into ten fractions. The LC-MS data were acquired at two facilities to assess inter-laboratory reproducibility. Both methods resulted in a high proteome coverage (>5,000 proteins) with low missing values on protein level (<2%) The TMT workflow led to 15-20% more identified proteins and a slightly better quantitative precision whereas the quantitative accuracy was better for the DIA method. The quantitative performance was benchmarked by the number of true positives (UPS2 proteins) within the top 100 candidates. TMT and DIA performed similar. The quantitative performance of the DIA data could be even improved by searching them directly against a database instead of using a project specific library. Our experiments also demonstrated that both methods can be easily transferred between facilities.
Project description:Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible and multiplexed quantification remains challenging especially for analysis of post-translational modifications (PTMs), such as phosphorylation. Here we compared the most popular quantification techniques for phosphoproteomics in context of cell-signaling studies: label-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS2- and MS3-measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide-ratios, we found LFQ and SILAC to be the most accurate techniques. MS2-based TMT suffered from substantial ratio compression, which MS3-based TMT could partly rescue. However, when analyzing phosphoproteome changes in the DNA damage response (DDR), we found that MS3-based TMT was outperformed by MS2-based TMT as it identified most significantly regulated phosphopeptides due to its higher precision and higher number of identifications. Finally, we show that the high accuracy of MS3-based TMT is crucial for determination of phosphorylation site stoichiometry using a novel multiplexing-dependent algorithm.