Chemical Tagging of Protein Lipoylation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Escherichia Coli Mus Musculus (mouse)
TISSUE(S): Macrophage
SUBMITTER: 
Qi Tang
LAB HEAD: Xing Chen
PROVIDER: PXD021948 | Pride | 2020-11-16
REPOSITORIES: Pride
Similar Datasets
Project description:Transcription factors play diverse roles during embryonic development, combinatorially controlling multiple cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholinos (cMOs) with photoactivatable fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies. As a proof of principle, we have dynamically profiled No tail-a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering and characterizing discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies demonstrate how optically controlled chemical tools can be use to probe developmental processes with spatiotemporal precision and reveal the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor. Zebrafish zygotes were injected with a mixture of ntla caged morpholino (cMO) and caged fluorescein dextran (cFD), and a 100 µm-diameter region of the shield was UV-irradiated at 6 hours post fertilization (hpf). The UV irradiation generated an active morpholino targeting the ntla 5'UTR and simultaneously labeled the cells with green fluorescence. By 36 hpf, the irradiated, green-fluorescent cells contributed to the medial floor plate rather than the notochord, consistent with earlier proposals that Ntla acts as a transcriptional switch between these two cell fates. Combining these caged reagents with microarray analysis identified transcriptional changes coincident with loss of ntla at 9 hpf and subsequent notochord fate respecification. Zebrafish zygotes were injected with a mixture of ntla cMO/cFD, and a 100 µm-diameter region of the shield was UV-irradiated at 6 hpf. Control embryos were injected with cFD and similarly irradiated at 6 hpf. Experimental and control sets of embryos were enzymatically dissociated at 9 hpf, green-fluorescent cells were isolated by FACS, and collected in trizol. Thirty embryos were used for each experiment or controls, each yielding approximately 8,000 green fluorescent cells. Five biological replicates were performed along with five paired controls. Total RNA was isolated from each sample, reverse transcribed, and amplified using WTA2 TransPlex Complete Whole Transcriptome Amplification kit (Sigma) using a miniaturized procedure and manufacturer-recommended incubation steps. The synthesized cDNA was sent to Nimblegen for dye labeling and hybridization. Samples were hybridized to the Nimblegen 2007 (Zv 7) Danio rerio Gene Expression Array chip using one dye per chip. Raw probe data (.pair file) was subjected to RMA, normalization, background correction, and generation of gene expression summary (.calls file) by Nimblegen. Processed data was analyzed in ArrayStar software (DNAStar). Significantly affected genes (fold change > 2 and p-value < 0.10) were identified using a moderated t-test.
2012-03-01 | E-GEOD-31880 | biostudies-arrayexpress
Project description:Transcription factors play diverse roles during embryonic development, combinatorially controlling multiple cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholinos (cMOs) with photoactivatable fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies. As a proof of principle, we have dynamically profiled No tail-a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering and characterizing discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies demonstrate how optically controlled chemical tools can be use to probe developmental processes with spatiotemporal precision and reveal the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor. Zebrafish zygotes were injected with a mixture of ntla caged morpholino (cMO) and caged fluorescein dextran (cFD), and a 100 µm-diameter region of the posterior chordamesoderm 125 μm anterior to the center of Kupffer’s vesicle was UV-irradiated at 12 hours post fertilization (hpf). The UV irradiation generated an active morpholino targeting the ntla 5'UTR and simultaneously labeled the cells with green fluorescence. By 36 hpf, the irradiated, green-fluorescent cells contributed to both notochord and floor plate, but the notochord cells were partially vacuolated and disorganized. Combining these caged reagents with microarray analysis identified transcriptional changes coincident with loss of ntla at 16 hpf and subsequent notochord differentiation. Zebrafish zygotes were injected with a mixture of ntla cMO/cFD, and a 100 µm-diameter region of the posterior chordamesoderm 125 μm anterior to the center of Kupffer’s vesicle was UV-irradiated at 12 hpf. Control embryos were injected with cFD and similarly irradiated at 12 hpf. Experimental and control sets of embryos were enzymatically dissociated at 16 hpf, green-fluorescent cells were isolated by FACS, and collected in trizol. Thirty embryos were used for each experiment or controls, each yielding approximately 8,000 green fluorescent cells. Five biological replicates were performed along with five paired controls. Total RNA was isolated from each sample, reverse transcribed, and amplified using WTA2 TransPlex Complete Whole Transcriptome Amplification kit (Sigma) using a miniaturized procedure and manufacturer-recommended incubation steps. The synthesized cDNA was sent to Nimblegen for dye labeling and hybridization. Samples were hybridized to the Nimblegen 2007 (Zv 7) Danio rerio Gene Expression Array chip using one dye per chip. Raw probe data (.pair file) was subjected to RMA, normalization, background correction, and generation of gene expression summary (.calls file) by Nimblegen. Processed data was analyzed in ArrayStar software (DNAStar). Significantly affected genes (fold change > 2 and p-value < 0.10) were identified using a moderated t-test.
2012-03-01 | E-GEOD-31881 | biostudies-arrayexpress
Project description:To identify cell type-specific RNA without cell sorting, mice expressing UPRT in a specific cell type were exposed to 4-thiouracil to label RNA only in the UPRT-expressing cells. Total RNA was extracted from tissues and treated with iodoacetamide followed by RNA-seq library preparation. The reads generated from 4-thiouracil-containing transcripts should contain T to C mismatches as a result of 4-thiouracil incorporations and thus can be identified as the transcripts generated in the UPRT-expressing cells.
2018-07-09 | E-MTAB-6353 | biostudies-arrayexpress
Project description:The aim of the project was to characterize changes in gene expression that are associated with induced autophagic flux. We engineered HeLa, HEK 293 and SH-SY5Y cell lines to express tandem-fluorecent LC3 (tf-LC3). The ratio of the red and green fluorophores indicated how much of the LC3 is in the acidic interior of lysosomes, which was a proxy measure for autophagic flux. RNA sequencing was performed for the cell lines at baseline and 1h, 15h and 30h after treatments. Additional untreated samples were also sequenced at some but not all time points. Autophagy was induced by amino acid starvation or mTOR inhibition (AZD8055).
2022-08-25 | E-MTAB-12020 | biostudies-arrayexpress
Project description:We describe a chemical method to label and purify 4-thiouridine (s4U) -containing RNA. We demonstrate that methanethiolsulfonate (MTS) reagents form disulfide bonds with s4U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s4U-labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA such as 4-thiouridine-tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover. s4U metabolic labeling of RNA in 293T cells, followed by biochemical enrichment of labeled RNA with two biotinylation reagents, RNAs >200nt and miRNAs in separate experiments
2015-09-03 | E-GEOD-60988 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE31880: Resolution of ntla-dependent transcriptome at 9 hpf using caged molecules GSE31881: Resolution of ntla-dependent transcriptome at 16 hpf using caged molecules Refer to individual Series
2012-03-01 | E-GEOD-31882 | biostudies-arrayexpress
Project description:One major knowledge gap in systems biology is the quantification of dynamic protein synthesis rates in response to cell perturbation. To address this gap, we have developed MITNCAT, multiplex isotope tagging / non-canonical amino acid tagging, a novel method enabling the robust quantification of proteome-wide protein synthesis rates with high temporal resolution (15-30 minutes) across multiple time points or treatment conditions, in a single analysis. MITNCAT combines multiplexed isobaric mass tagging with bioorthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized proteins with azidohomoalanine (Aha) and pulsed SILAC (pSILAC), thus providing enrichment and multiplexed quantification of newly synthesized proteins. We demonstrate the application of MITNCAT to quantify altered protein synthesis rates following induction of the unfolded protein response (UPR) and epidermal growth factor (EGF) stimulation. Eliciting the UPR by blocking N-glycosylation results in a global down-regulation of protein synthesis, with stronger down-regulation of proteins involved in the glycolysis pathway and protein synthesis machinery (ribosomal proteins, initiation factors, and elongation factors), but up-regulation of several key protein-folding chaperones. MITNCAT enables the quantification of waves of temporally distinct protein synthesis in response to EGF stimulation, with altered protein synthesis on dozens of proteins detectable in the first 15 minutes. Comparison of protein synthesis with RNA sequencing and ribosome footprinting allowed the distinction between protein synthesis driven by an increase in transcription versus that driven by an increase in translational efficiency. Temporal delays between ribosome occupancy and protein synthesis were observed and found to correlate with altered codon usage in significantly delayed proteins.
2018-12-04 | PXD009592 | Pride
Project description:Efficient methods to introduce bioorthogonal groups, such as terminal alkynes, into biomolecules are important tools for chemical biology. State-of-the-art approaches are based on the introduction of a linker between the targeted amino acid and the alkyne, and still present limitations of either reactivity, selectivity or adduct stability. Herein, we present a new ethynylation method of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. In contrast to other approaches, the acetylene group is directly introduced onto the thiol group of cysteine and can be used in one-pot in a copper-catalyzed alkyne-azide cycloaddition (CuAAC) for further functionalization. Labeling proceeded with reaction rates comparable or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab. Under optimized conditions, high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed a much-improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine-reagent based probes. Fine-tuning of the EBX reagents allowed optimization of their reactivity and physical properties for the desired application.
2020-04-07 | PXD018165 | Pride
Project description:E. coli cultures were exposed to tellurite 0.5 µg/ml during 15 min. Total RNA was extracted and cDNA labeled probes were generated by reverse transcription using Alexa 555 and Alexa 647 fluorophores. These probes were used to hybridize genomic slides containing genomic arrays to determine global transcriptional changes. Two-conditions experiment, antibacterial vs. Untreated control cells. Biological replicates: 2 control, 2 toxicants exposed cells, independently grown and harvested. One replicate per array. Dye swap conditions.
2014-07-30 | E-GEOD-58176 | biostudies-arrayexpress
Project description:ATAC-seq was performed to map changes in chromatin accessibility in monocytes during in vitro differentiation. In addition to control cells, we also studied the impact of siRNA mediated knock-down of key transcription factors on accessible chromatin in monocyte-derived dendritic cells.
2021-02-03 | E-MTAB-9926 | biostudies-arrayexpress