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A phase-separated CO2-fixing pyrenoid proteome determined by TurboID in Chlamydomonas reinhardtii

ABSTRACT: Mass spectrometry data generated from TurboID-based proximity labeling of the Chlamydomonas phase-separated pyrenoid

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Chlamydomonas Reinhardtii (ncbitaxon:3055)

SUBMITTER: Luke Mackinder

PROVIDER: MSV000091853 | MassIVE | Thu May 04 00:51:00 BST 2023

SECONDARY ACCESSION(S): PXD041970

REPOSITORIES: MassIVE

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TurboID-mediated proximity labeling for screening interacting proteins of FIP37 in Arabidopsis

Project description:Proximity labeling (PL) was recently developed to detect protein–protein interactions (PPIs) and members of subcellular multiprotein structures in living cells. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity, such as biotin ligase, to a protein of interest (bait protein) to biotinylate adjacent proteins. The biotinylated protein can be purified by strep-tavidin beads, and identified by mass spectrometry (MS). TurboID is an engineered biotin ligase with high catalytic efficiency, which is used for proximity labeling. Although TurboID-based proximity labeling technology has been successfully established in mammals, its application in plant systems is limited. Here, we report the usage of TurboID for proximity labeling of FIP37, a core member of m6A methyltransferase complex, to identify FIP37 interacting proteins in Ara-bidopsis thaliana. By analyzing the MS data, we found 214 proteins biotinylated by GFP-TurboID-FIP37 fusion, including five components of m6A methyltransferase complex that have been previously confirmed. Therefore, the identified proteins may include potential proteins directly involved in the m6A pathway or functionally related to m6A-coupled mRNA processing due to spatial proximity. Moreover, we demonstrated the feasibility of proximity labeling tech-nology in plant epitranscriptomics studies, thereby expanding the application of this technology to more subjects of plant research.

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Interactome analysis of C. elegans synapses by TurboID-based proximity labeling

Project description:Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. Using the highly active biotin ligase TurboID, we optimize a proximity labeling protocol for C. elegans. We use this protocol to characterise the proteomes of the worm’s gut, muscle, skin, and nervous system. We express TurboID exclusively in the pair of AFD neurons and show we can identify known and previously unknown proteins expressed selectively in AFD. We knock TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identify many known ELKS-1 interacting proteins as well as previously uncharacterised synaptic proteins. Versatile vectors, and the inherent advantage of C. elegans for biochemistry, make proximity labeling a valuable addition to the nematode’s armory.

2021-09-15 | PXD027068 | Pride

TurboID-based proximity labeling to identify LONP1-targeting proteins

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Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines.

Project description:Different brain cell types play distinct roles in brain development and disease via cellular mechanisms. Molecular characterization of these cellular mechanisms using cell type-specific approaches, particularly at the protein (proteomic) level, can provide biological and therapeutic insights. Conventional approaches to investigate cell type-specific proteomes from brain pose several technical barriers. To overcome these, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using TurboID with a Nuclear Export Sequence (TurboID-NES), coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy to sample cell type-specific proteomes in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID-NES approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. We generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing TurboID-NES to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-NES expression and biotinylation did not significantly impact homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration of BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. TurboID-NES mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under resting conditions. Acute LPS treatment significantly altered microglial proteomes, but not N2A proteomes, and the LPS effect was partly captured by analysis of the TurboID-NES-labeled proteome of BV2 cells.

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PML nuclear bodies form a regulatory hub via liquid-liquid phase separation for TRIM33 control of Lefty1/2 genes in mouse embryonic stem cells [CHIP-seq]

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Project description:Proximity editing to identify RNA in phase separated RBPs condensates

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2023-01-10 | PXD037432 | Pride

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