Project description:Pulse (P) - Chase (C) SILAC analysis of mitochondrial complex assembly
Bogenhagen et al (2018) Cell Reports
Pulse-chase times in hours (P#C#)
Project description:Pulse chase SILAC was used to identify protein turnover within human macrophages infected with mycobacterium tuberculosis CDC1551, a ppe38-71 mutant strain, a complemented strain and an uninfected control.
Project description:Manassantin A is a natural product that has been isolated from the perennial herb Saururus chinensis Baill and the aquatic plant Saururus cernuus. Manassantin A has been shown to possess potent hypoxia inducible factor 1 alpha (HIF-1α) inhibitory activity in a cell-based assay screen of thousands of natural products. Manassantin A holds promise as an anti-cancer drug since it has been shown to selectively target tumor cells over normal cells. Due to the complex biological pathways involved in cancer and hypoxia, it is difficult to determine the mode-of-action by which manassantin A inhibits HIF-1. While some of the biological activities of manassantin A have been discovered in various cell-based activity assays, the molecular basis of manassantin A’s biological activities is not well characterized. The proteins in a hypoxic MDA-MB-231 cell lysate were screened for interactions with manassantin A using large scale experiments to uncover novel manassantin A interactions that lead to the drug’s HIF-1 inhibition and anti-cancer activity. Two energetics-based approaches were utilized in this manassantin A mode-of-action study: iTRAQ-SPROX and SILAC-Pulse Proteolysis. In these energetics-based approaches, protein stability is measured using the chemical denaturant dependence of either a methionine oxidation reaction (iTRAQ-SPROX) or a thermolysin protease digest (SILAC-Pulse Proteolysis). Using boh of these techniques, the stability of proteins in the absence and presence of excess manassantin A was monitored to assess ligand-induced protein stability changes.
Project description:We developed a simple method that combined biochemical mitochondria isolation and pulse SILAC approach to monitor the mitochondrial translation. Our approach allows us to quantify 12 out of the 13 mitochondrial translation products, the highest coverage among analogous methods reported, and provide a global picture of (post-)translational regulation in mitochondria. Replicate 1: H-CRP_M-DMSO, Replicate 2: M-CRP_H-DMSO.
Project description:Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation (MT) control, it is challenging to identify and quantify the mitochondrial-encoded proteins because of their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture. Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of oxidative phosphorylation complexes. We found that inhibition of MT led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study MT programs in many contexts, including oxidative stress and mitochondrial disease.
Project description:To measure translational efficiency in FMRP depletion, we purified RNAs from either wild-type or FMR1-knockout (FMR1-KO) SH-SY5Y cells generated for SILAC coupled to LC-MS/MS analysis and performed RNA-seq to quantitate mRNA abundance to normalize their protein abundance.
Project description:Cells were grown in light (R0K0) SILAC media (AthenaES) for 7 days, they were then incubated in light media for 24 hours in respective condition (normoxia neutral (NN) pH: 7.4; hypoxia neutral (HN)1% O2, pH: 7.4; hypoxia acidosis (HA), 1% O2, pH=6) and pulsed with heavy (R10K8) SILAC media (AthenaES) for 16 hr (TMT-pSILAC) following treatment.
Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase. An RNA stablity experiment design type examines stability and/or decay of RNA transcripts. Keywords: RNA_stability_design