Project description:Proteome analysis of Mouse Embryonic Fibroblasts (MEFs) cells with different ras mutations display distinct phenotypes
-Legend File : GIL refers to Q61L
Project description:In molecular biology, the design of mechanistic experiments has to be optimized by considering statistical and biological principles. In contrast to statistical principles, biological principles of experimental design are not universally formulated. In an attempt to pinpoint generally acceptable rules, we investigated the importance of determining the optimal ranges of scale of i.e. dose and time in gene expression experiments. We propose a protocol for executing small scale, genome wide, range finding studies, covering a wide range of the potentially relevant part of the design space to find the optimal ranges of experimentation. This protocol is executed and a proof-of-concept is presented, where this approach is tested for both an in-vitro and an in-vivo study that aim to unravel DNA repair mechanisms provoked after UV radiation. We identified four challenges of range finding studies in omics experimentation; (1) the modularity of biological processes, (2) their dynamics, (3) the extent to which end-points indicate biological processes, and (4) the costs associated with the assays, which are all addressed by our approach. 57 skin biopt samples taken from 12 individual mice on 8 timepionts and for 6 different UV-B doses. Per mouse 5 skin biopts were samples in time
Project description:Fibroblasts can be directly reprogrammed to induced renal tubular epithelial cells (iRECs) using four transcription factors. These engineered cells may be used for disease modeling, cell replacement therapy or drug and toxicity testing. Direct reprogramming induces drastic changes in the transcriptional landscape, protein expression, morphological and functional properties of cells. However, how the metabolome is changed by reprogramming and to what degree it resembles the target cell type remains unknown. Using untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-MS, we characterized the metabolome of mouse embryonic fibroblasts (MEFs), iRECs, mIMCD-3 cells, and whole kidneys. Metabolic fingerprinting can distinguish each cell type reliably, revealing iRECs are most similar to mIMCD-3 cells and clearly separate from MEFs used for reprogramming. Treatment with the cytotoxic drug cisplatin induced typical changes in the metabolic profile of iRECs commonly occurring in acute renal injury. Interestingly, metabolites in the medium of iRECs, but not of mIMCD-3 cells or fibroblast could distinguish treated and non-treated cells by cluster analysis. In conclusion, direct reprogramming of fibroblasts into renal tubular epithelial cells strongly influences the metabolome of engineered cells, suggesting that metabolic profiling may aid in establishing iRECs as in vitro models for nephrotoxicity testing in the future.
Project description:In molecular biology, the design of mechanistic experiments has to be optimized by considering statistical and biological principles. In contrast to statistical principles, biological principles of experimental design are not universally formulated. In an attempt to pinpoint generally acceptable rules, we investigated the importance of determining the optimal ranges of scale of i.e. dose and time in gene expression experiments. We propose a protocol for executing small scale, genome wide, range finding studies, covering a wide range of the potentially relevant part of the design space to find the optimal ranges of experimentation. This protocol is executed and a proof-of-concept is presented, where this approach is tested for both an in-vitro and an in-vivo study that aim to unravel DNA repair mechanisms provoked after UV radiation. We identified four challenges of range finding studies in omics experimentation; (1) the modularity of biological processes, (2) their dynamics, (3) the extent to which end-points indicate biological processes, and (4) the costs associated with the assays, which are all addressed by our approach. 48 MEF samples having various combinations of 9 timepoints and 6 UV-C Doses without replication were used
Project description:This project have LC-MSMS analysis results of label free quantitation of N-terminome enrichment of mouse proteome. N-terminal modification of mouse embryonic fibroblasts (MEFs) lacking Naa10 show negligible difference between wild-type and Naa10 mutant.