Dataset Information

‘Off the shelf’ proximity biotinylation for interaction proteomics

ABSTRACT: Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins of interest using an endogenous antibody against the bait protein. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, the histone modification H3K9me3 and the chromatin remodeler BRG1. Amongst the main findings, our experiments revealed Flywch1 as an essential protein that interacts with H3K9me3-marked centromeric heterochromatin. The ProtA-Turbo enzyme represents an ‘off the shelf’ proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.

INSTRUMENT(S): Orbitrap Fusion, Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Primary Cell, Permanent Cell Line Cell, Cell Culture, Fibroblast

SUBMITTER: Irene Santos Barriopedro

LAB HEAD: Professor Michiel Vermeulen

PROVIDER: PXD025012 | Pride | 2021-07-12

REPOSITORIES: Pride

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Publications

Off-the-shelf proximity biotinylation for interaction proteomics.

Santos-Barriopedro Irene I van Mierlo Guido G Vermeulen Michiel M

Nature communications 20210818 1

Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysa ...[more]

PMID: 34408139

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Project description:Patients received standardized coartem therapy. Briefly, each pill contains 20 mg of artemether and 120 mg of lumefantrine. The dose and total coarse of tablets is based on the patients body weight. Blood was drawn from patients prior to anti-malarial treatment (day 0) and after treatment (day42 for children and day 28 for adults). Peripheral Blood Mononuclear Cells were isolated from all patients. Briefly, for PBMC isolation, whole blood from was diluted 1:2 in sterile PBS and overlaid on Ficoll Paque-PLUS in 50 mL conical tubes and centrifuged 800xg for 15 minutes at 25°C with no brake. Resulting buffy coats were collected and washed twice in RPMI 1640 medium, and then cells were treated with Red Blood Cell Lysis Buffer for 3 minutes at room temperature. Cells were washed in RPMI and counted on a hemacytometer. Monocytes were purified from PBMC using a miltenyi Pan Monocyte Isolation negative selection kit according to the manufacturer's instructions. For each sample. 50,000 viable cells were pelleted and lysed with cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and incubated in ice for 3 minutes. The lysaste was washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin. Nuclei were pelleted at 500 RCF for 10 min at 4 degrees celcius. The supernatant was discarded and the pellet was resuspended in 50 uL of transposition mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS,0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O). The reaction was incubated for 30 minutes in a thermomixer with 1000 RPM mixing. Zymo DNA Clean and concentrator kit (cat# D4014) was used as a cleanup step. The DNA was amplified at 98 degrees C for 30 seconds, then 13 cycles of: 98 degrees C for 10 seconds, 63 degrees C for 30 seconds, 72 degrees C for 1 minute.The library was evaluated in an Agilent TapeStation system and the DNA concentration evaluated by Qubit. Sequencing was performed on Illumina Nextseq 500 with Buffer cartridge v2: Ref: 15057941, High output reagent cartridge v2: Ref: 15057934, NextSeq accessory Box v2: Ref: 15058251, and High output Flow Cell cartridge v2.5: Ref: 20022408

Dataset Information

‘Off the shelf’ proximity biotinylation for interaction proteomics

Publications

Off-the-shelf proximity biotinylation for interaction proteomics.

Similar Datasets

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