ABSTRACT: Biotinylated diUb species and monoubiquitin were immobilized on streptavidin-coated magnetic beads and subjected to an in vitro pulldown assay using HCT116 whole-cell extracts.
Project description:GST pull-down assay using crude extracts from HCT116 cells to analyze the selectivity of the UFD1-NBM peptide in a cellular context.
Project description:To identify an uncharacterized endogenous defective protein pool, we immunoprecipitated Flag-tagged TanGIBLE from HeLa cell extracts, and co-immunoprecipitated proteins werer subjected to LC-MS/MS analysis.
Project description:In mammals, loss of food intake and reduced mechanical loading/activity of skeletal muscles leads to a very rapid loss in mass and function. However, during hibernation in bears, despite spending months without feeding and with very modest muscle activity, only moderate muscle wasting is observed. Part of this tissue sparing is due to a highly reduced metabolic activity in almost all tissues, including skeletal muscle. Interestingly, myosin, one of the most abundant proteins in skeletal muscle, can have different metabolic activities in inactive muscle. Therefore, to evaluate the functional and metabolic alterations in hibernating muscles, we performed an analysis on a single muscle fiber level. Individual fibers were taken from biopsies of the same bears either during hibernation or during the active phase in the summer. We confirm that muscle fibers from hibernating bears show no loss of fiber size and a mild reduction in force generating capacity. However, ATPase activity of single muscle fibers taken from hibernating bears show a significant reduction in ATPase activity, which is due to a reduced ATP turnover by myosin. By performing a single fiber proteomics analysis, we could determine in a fiber type specific manner that muscle fibers undergo a major remodeling of their proteome. Both type 2A and type 1/2A mixed fibers show a marked reduction in mitochondrial proteins during hibernation, with a decrease in proteins linked to the TCA cycle and mitochondrial translation.
Project description:Novel development makes remote real-time analysis with possible translation to in-vivo a reality. Remote Infrared Matrix Assisted Laser Desorption Ionization (Remote IR MALDI) system with endogenous water as matrix becomes real and allows to envisage real-time proteomics to be performed in the in-vivo context. Remote IR MALDI is demonstrated to be used to analyze peptides and proteins. Very interestingly, the corresponding mass spectra show ESI like charge states distribution, opening many applications for structural elucidation to be performed in real-time by Top-Down analysis. The charge states show no dependence toward laser wavelength or length of the transfer tube allowing for remote analyses to be perform 5 m away from the mass spectrometry (MS) instrument without modification of spectra. This brings also interesting features to the understanding of IR MALDI ionization mechanism
Project description:Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample-preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 (OT-2) robot that combines sample digestion, cleanup and Evotip loading in a fully automated manner, allowing the processing of up to 192 samples in 6 hours. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability even at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic Ti-IMAC beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.
Project description:Investigation of differentially expressed genes in human HCT116 cells after knockdown of FBXO28 for 16h and 36h. FBXO28 knockdown and control HCT116 cells. 4 replicates per time point (16h, 36h), including dye swaps.
Project description:Homologous sets of transcription factors direct conserved tissue-specific transcription, yet transcription factor binding events diverge rapidly between closely related species. We used hepatocytes from a Down syndrome mouse model containing human chromosome 21 to determine whether human genetic sequence or mouse nuclear environment primarily determines tissue-specific transcriptional regulation. Virtually all transcription factor binding locations, transcription initiation events and the resulting gene expression observed in human hepatocytes are recapitulated across the entire human chromosome 21 in the mouse nucleus. Thus, in homologous tissues, genetic sequence is largely responsible for directing transcriptional programs, and interspecies differences in epigenetics, cellular environment, and transcription factors themselves play secondary roles.
Project description:Embryonic stem cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2 and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal (Boyer et al. 2005; Loh et al. 2006; Chambers et al. 2003; Mitsui et al. 2003; Nichols et al. 1998). How external signals connect to this regulatory circuitry to influence embryonic stem cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in embryonic stem cells, the transcription factor Tcf3, co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog and other pluripotency factors and enhance pluripotency and self-renewal. Our results reveal that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation.