Identification of newly synthesized proteins in microbial communities using BONCAT and click chemistry
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ABSTRACT:
PROVIDER: PRJEB70937 | ENA |
REPOSITORIES: ENA
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Project description:Metaproteomics enables the description of microbial communities (MC). Microbial adaptation to changing environments is conducted by expressing newly synthesized proteins (nP) that can be difficult to distinguish from background proteins. Focusing on nP would add a new dimension to the metaproteomics of MC. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising approach to label nP without significantly influencing the natural behavior of MC. However, direct detection of the BONCAT-labeled nP is limited due to their low abundance compared to total protein. Consequently, enrichment of the BONCAT-labeled nP is essential. We present a workflow using click chemistry (CC) and affinity chromatography to isolate nP from MC. The workflow was developed using a mixture of E. coli (labeled) and yeast (unlabeled control) as a test system. The established workflow was also applied to an MC of a laboratory biogas reactor (LBR).
2024-10-17 | PXD047252 | Pride
Project description:Development of an improved workflow for newly synthesized proteome analysis, based on combined pulsed metabolic labelling with L-azidohomoalanine (AHA) and semi-automated click chemistry-based enrichments with magnetic alkyne agarose beads.
2023-10-19 | PXD036886 | Pride
Project description:The success of targeted therapies hinges on our ability to understand the molecular and cellular mechanism of action of these agents. Here we modify various BET bromodomain inhibitors, an exemplar novel targeted therapy, to create functionally conserved compounds that are amenable to click-chemistry and can be used as molecular probes in vitro and in vivo. Using click-proteomics and click-sequencing we provide new mechanistic insights to explain the gene regulatory function of BRD4 and the transcriptional changes invoked by BET inhibitors. In mouse models of acute leukaemia, we use high resolution microscopy and flow cytometry to highlight the underappreciated heterogeneity of drug activity within tumour cells located in different tissue compartments. We also demonstrate the differential distribution and effects of the drug in normal and malignant cells in vivo. These data provide critical insights that reveal the cellular and molecular details for the efficacy and limitations of these agents. This study provides a framework for the pre-clinical assessment of other conventional and targeted therapies.
2017-07-13 | GSE88749 | GEO
Project description:The success of targeted therapies hinges on our ability to understand the molecular and cellular mechanism of action of these agents. Here we modify various BET bromodomain inhibitors, an exemplar novel targeted therapy, to create functionally conserved compounds that are amenable to click-chemistry and can be used as molecular probes in vitro and in vivo. Using click-proteomics and click-sequencing we provide new mechanistic insights to explain the gene regulatory function of BRD4 and the transcriptional changes invoked by BET inhibitors. In mouse models of acute leukaemia, we use high resolution microscopy and flow cytometry to highlight the underappreciated heterogeneity of drug activity within tumour cells located in different tissue compartments. We also demonstrate the differential distribution and effects of the drug in normal and malignant cells in vivo. These data provide critical insights that reveal the cellular and molecular details for the efficacy and limitations of these agents. This study provides a framework for the pre-clinical assessment of other conventional and targeted therapies.
2017-07-13 | GSE88748 | GEO
Project description:Proteins that were newly-synthesized during ex vivo lung perfusion were labeled with an azido-sugar Ac4GalNAz in the perfusate. These proteins were enriched using click chemistry to attach an alkyne-desthiobiotin group to the azido-labeled proteins, then pulled out of solution using streptavidin beads. The overall goal is to better understand the effects of warm ischemia injury in lungs destined for lung transplantation.
2022-10-07 | MSV000090491 | MassIVE
Project description:The success of targeted therapies hinges on our ability to understand the molecular and cellular mechanism of action of these agents. Here we modify various BET bromodomain inhibitors, an exemplar novel targeted therapy, to create functionally conserved compounds that are amenable to click-chemistry and can be used as molecular probes in vitro and in vivo. Using click-proteomics and click-sequencing we provide new mechanistic insights to explain the gene regulatory function of BRD4 and the transcriptional changes invoked by BET inhibitors. In mouse models of acute leukaemia, we use high resolution microscopy and flow cytometry to highlight the underappreciated heterogeneity of drug activity within tumour cells located in different tissue compartments. We also demonstrate the differential distribution and effects of the drug in normal and malignant cells in vivo. These data provide critical insights that reveal the cellular and molecular details for the efficacy and limitations of these agents. This study provides a framework for the pre-clinical assessment of other conventional and targeted therapies.
2017-07-13 | GSE88750 | GEO
Project description:The success of targeted therapies hinges on our ability to understand the molecular and cellular mechanism of action of these agents. Here we modify various BET bromodomain inhibitors, an exemplar novel targeted therapy, to create functionally conserved compounds that are amenable to click-chemistry and can be used as molecular probes in vitro and in vivo. Using click-proteomics and click-sequencing we provide new mechanistic insights to explain the gene regulatory function of BRD4 and the transcriptional changes invoked by BET inhibitors. In mouse models of acute leukaemia, we use high resolution microscopy and flow cytometry to highlight the underappreciated heterogeneity of drug activity within tumour cells located in different tissue compartments. We also demonstrate the differential distribution and effects of the drug in normal and malignant cells in vivo. These data provide critical insights that reveal the cellular and molecular details for the efficacy and limitations of these agents. This study provides a framework for the pre-clinical assessment of other conventional and targeted therapies.
2017-07-13 | GSE88746 | GEO
Project description:The success of targeted therapies hinges on our ability to understand the molecular and cellular mechanism of action of these agents. Here we modify various BET bromodomain inhibitors, an exemplar novel targeted therapy, to create functionally conserved compounds that are amenable to click-chemistry and can be used as molecular probes in vitro and in vivo. Using click-proteomics and click-sequencing we provide new mechanistic insights to explain the gene regulatory function of BRD4 and the transcriptional changes invoked by BET inhibitors. In mouse models of acute leukaemia, we use high resolution microscopy and flow cytometry to highlight the underappreciated heterogeneity of drug activity within tumour cells located in different tissue compartments. We also demonstrate the differential distribution and effects of the drug in normal and malignant cells in vivo. These data provide critical insights that reveal the cellular and molecular details for the efficacy and limitations of these agents. This study provides a framework for the pre-clinical assessment of other conventional and targeted therapies.
2017-07-13 | GSE88747 | GEO
Project description:The analysis of the secretome provides important information on cellular communication and on the recruitment and behavior of cells in specific tissues. Especially in the context of tumors, knowledge on the composition of the secretome plays an important role for diagnosis and therapy. Mass spectrometry-based analysis of cell-conditioned media is widely used for the unbiased characterization of cancer secretomes in vitro. Azide-containing amino acid labeling in combination with click chemistry now facilitates this type of analysis without serum starvation. The modified amino acids, however, are less efficiently incorporated into newly synthesized proteins. Utilizing transcriptome and proteome analysis, we describe here in detail the effects of labeling cells with the methionine analog azidohomoalanin (AHA) on the modulation of gene expression and protein composition. We observe that 11% – 34% of the proteins detected in the secretome were affected by AHA labeling regarding their expression levels in the transcriptome and/or proteome. Performing gene ontology analyses, our results provide a detailed view on the AHA-based induction of cellular stress and apoptosis-related pathways and provide first insights on how this might affect the composition of the secretome on a global scale.
2023-07-05 | GSE211231 | GEO
Project description:Bioorthogonal chemistry introduces affinity-labels into biomolecules with minimal disruption to the original system and is widely applicable in a range of contexts. In proteomics, immobilized metal affinity chromatography (IMAC) enables enrichment of phosphopeptides with extreme sensitivity and selectivity. Here, we adapt and combine these superb assets in a new enrichment strategy using phosphonate-handles, which we term ‘PhosID’. In this approach, ‘click-able’ phosphonate-handles are introduced into proteins via 1,3-dipolar Huisgen-cycloaddition to azido-homo-alanine (AHA) and IMAC is then used to enrich exclusively for phosphonate-labeled peptides. In interferon-gamma (IFNγ) stimulated cells, PhosID enabled the identification of a large number of IFN responsive newly synthesized proteins (NSPs) whereby we monitored the differential synthesis of these proteins over time. Collectively, these data validate the excellent performance of PhosID with efficient analysis and quantification of hundreds of NSPs by single LC-MS/MS runs. We envision PhosID as an attractive and alternative tool for studying stimuli-sensitive proteome subsets.
2020-07-28 | PXD018272 | Pride