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N Gene TurboID (Group2 +p50)

ABSTRACT: Nicotiana benthamiana plants were infiltrated with three different constructs: 1) N-TurboID-3HA 2) NTIR-TurboID-3HA or 3) Citrine-TurboID-3HA. N-TurboID is the full length N-gene fused to TurboID, NTIR-Turbo is the TIR domain of the N-gene fused to turboID, and Citrine-TurboID is the control construct. Three biological replicates for each construct were labeled with TMT10Plex reagents (Lot TB260979). N-TurboID (TMT: 126, 127N, 126C); NTIR-TurboID (TMT: 128N, 128C, 129N); citrine-TurboID (TMT: 129C, 130N, 130C). Biotinolayted proteins were enriched with streptavidin beads (equal protein input onto beads) and digested into peptides with Trypsin/LysC. An equal volume of peptides were labeled with TMT reagents.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Nicotiana Benthamiana (ncbitaxon:4100)

SUBMITTER: Justin Walley

PROVIDER: MSV000083019 | MassIVE | Wed Oct 10 09:00:00 BST 2018

REPOSITORIES: MassIVE

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Publications

TurboID-based proximity labeling reveals that UBR7 is a regulator of N NLR immune receptor-mediated immunity.

Zhang Yongliang Y Song Gaoyuan G Lal Neeraj K NK Nagalakshmi Ugrappa U Li Yuanyuan Y Zheng Wenjie W Huang Pin-Jui PJ Branon Tess C TC Ting Alice Y AY Walley Justin W JW Dinesh-Kumar Savithramma P SP

Nature communications 20190719 1

Nucleotide-binding leucine-rich repeat (NLR) immune receptors play a critical role in defence against pathogens in plants and animals. However, we know very little about NLR-interacting proteins and the mechanisms that regulate NLR levels. Here, we used proximity labeling (PL) to identify the proteome proximal to N, which is an NLR that confers resistance to Tobacco mosaic virus (TMV). Evaluation of different PL methods indicated that TurboID-based PL provides more efficient levels of biotinylat ...[more]

PMID: 31324801

Similar Datasets

N Gene TurboID (Group1 -p50)

Project description:Nicotiana benthamiana plants were infiltrated with three different constructs: 1) N-TurboID-3HA 2) NTIR-TurboID-3HA or 3) Citrine-TurboID-3HA. N-TurboID is the full length N-gene fused to TurboID, NTIR-Turbo is the TIR domain of the N-gene fused to turboID, and Citrine-TurboID is the control construct. Three biological replicates for each construct were labeled with TMT10Plex reagents (Lot TB260979). N-TurboID (TMT: 126, 127N, 126C); NTIR-TurboID (TMT: 128N, 128C, 129N); citrine-TurboID (TMT: 129C, 130N, 130C). Biotinolayted proteins were enriched with streptavidin beads (equal protein input onto beads) and digested into peptides with Trypsin/LysC. An equal volume of peptides were labeled with TMT reagents.

2018-10-10 | MSV000083018 | MassIVE

Identification of myelin basic protein interactome using TurboID proximity labeling proteomics

Project description:The protein environment of myelin basic protein (MBP), its uncharged form, and cyclindependent kinase inhibitor 1 (p21Cip1) were analyzed using two different approaches to identify protein partners thereof. The first one was a state-of-the-art enzymatic approach (TurboID), which utilizes an engineered biotin ligase that uses ATP to convert biotin to reactive biotin-AMP that covalently attaches to nearby proteins, followed by enrichment of biotin-labeled proteins on streptavidin beads and MS analysis of the resulting protein pool. Classical immunoprecipitation with Flag-tag fused to the target protein in combination with MS analysis was used as a reference method. As a result, two pools of potential interactors of MBP and uncharged MBP were identified. It was shown that TurboID, although giving a high level of background biotinylation, is a more reproducible technique compared to the immunoprecipitation, possibly due to higher affinity during the fraction enrichment process and easier maintaining of uniform conditions for the process, as well as interaction conditions being closer to in vivo conditions.

2023-01-25 | PXD039467 | Pride

Chicken Tandem-Mass-Tag quantitative mass spectrometry analysis

Project description:About “MS210144-1 to MS210144-12”: 6 samples: cell body-1#, cell body-2#, cell body-3#, Migrasome-1#, Migrasome-2#, Migrasome-3#, were labeled with tandem mass tags (TMT) reagents and then mixed. These combined TMT-labeled peptides were separated into 12 fractions (shown in list: from MS210144-1 to MS210144-12), then desalted by Sep-Pak columns and separated with a UPLC 3000 system and with an XBridgeTM BEH300 C18 column.

2022-10-12 | PXD036647 | Pride

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in hematopoietic cells

Project description:The global protein interactions of the oncofusion protein PML::RARA were identified using proximity labeling followed by mass spectrometry. Briefly, Lineage-depleted bone marrow cells from C57Bl/6 mice were transduced with MSCV-IRES-GFP retroviruses containing an enhanced biotin ligase (TurboID) fused to either the N-terminus or C-terminus of PML::RARAWT (TurboID-PML::RARAWT and PML::RARAWT-TurboID respectively), TurboID-PML::RARAC88A (mutation in the DNA binding domain of RARA), or a TurboID cDNA alone. A flexible Glycine/Serine linker placed between TurboID and PML::RARA allows TurboID to freely rotate, and biotinylate proteins within 10 angstroms of the TurboID-PML::RARA fusion protein. Four days following the first round of transduction, cells were flow sorted on GFP+ cells. Two days following the sort, 1-5 million cells were treated with 50 uM Biotin for four hours at 37 degrees C. Cell lysates were prepared, sonicated, and incubated with streptavidin agarose beads overnight. Beads were washed, peptides were eluted and digested, and then run on a timsTOF Pro 2.

2023-08-23 | MSV000092739 | MassIVE

Microarray analysis of the effects of a Î³-tocopherol-rich mixture of tocopherols on global gene expression in the prostates of TRAMP mice

Project description:We used whole mouse genome microarrays to the effects of Î³-TmT on global gene expression in the prostate TRAMP and age-matched C57BL/6 mice were administered either 600 mg/kg of Î³-TmT or a control vehicle via oral gavage. Twelve hours after dosing, prostate tissues were collected for RNA extraction.

2014-09-16 | E-GEOD-61416 | biostudies-arrayexpress

An atlas of the aging mouse transcriptome and proteome reveals the features of age-related post-transcriptional dysregulation

Project description:We've collected bulk RNA-Seq and TMT-labeled proteomic data of aorta, brain, heart, kidney, liver, lung, muscle (gastrocnemius muscle), and skin of 6-, 15-, 24-, and 30-months old male C57BL/6J mice. The tissues used for transcriptomic analysis and proteomic analysis were collected from the same mice. All analyses were conducted with 4 biological replicates.

2024-09-18 | GSE225576 | GEO

Germination and appressorium formation in Magnaporthe grisea

Project description:This series examines differential gene expression during germination and appressorium formation by the rice blast fungus Magnaporthe grisea. Conidia were germinated on either the hydrophobic (inductive) or hydrophilic (non-inductive) side of GelBond. RNA was extracted from conidia and following incubation for 7 and 12 hours. RNA from two biological replications of each treatment was pooled in equal amounts and labeled with both cy3 and cy5 dyes using the Agilent Technologies Low Input Linear Amplification Kit. Hybridizations were performed on Agilent Technologies whole genome oligo rice blast arrays (product G4137A) using manufacturer prescribed protocols and reagents in an interlaced loop design in which each treatment was paired with every other. This series contains a total of 10 hybridizations; each treatment was used in 4 hybridizations (2 with cy3 and 2 cy5). Spot fluorescence was normalized using Lowess within and between microarray slides in Bioconductor and gene expression profiles analyzed in GeneSpring. Keywords: other

2004-11-10 | GSE1945 | GEO

proteomics analysis of senescence cells with drug

Project description:Trypsin-digested peptides were labeled using 10-plex TMT reagent (Thermo Scientific, Rockford, IL). Peptide samples (50 ug each) were labeled TMT reagents. For proteome analysis, LC-MS/MS analysis was performed using an EASY-nLC 1200 UPLC (Thermo Scientific) coupled to a Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific).

2022-08-22 | MSV000090180 | MassIVE

Comparison of two A. nidulans strains: control CV125 and transgenic NA1363 harboring E. coli LacZ transgene.

Project description:Transcriptional profiling of two A. nidulans strains, aiming to compare the control strain CV125 (pabaA1) with the transgenic strain NA1363 (pabaA1) harbouring a gpdAp-lacZ-trpCt.riboB construct integrated in chromosome VIII. This construct, carried out in a derivative of the pBR322 vector, contained the E. coli lacZ sequence (encoding ß-galactosidase) fused to the A. nidulans trpC terminator and placed under the control of the strong and constitutive promoter of the A. nidulans gpdA gene, and the riboB gene as a selection marker.

2016-04-12 | GSE76918 | GEO

Single cell analysis of macrophages exposed to beads coated with LPS from Salmonella enterica subsp. typhimurium

Project description:We present a detailed single cell analysis of the macrophage response to LPS from Salmonella enterica. By combining single cell transcriptional analysis, fluorescently labeled, LPS-coated beads, and cytometry we are able to distinguish the responses of macrophages that have internalized LPS-coated beads and those that have not.

2015-09-07 | GSE65529 | GEO

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