Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Virotrap: Abducting Protein Complexes from Mammalian Cells

ABSTRACT: Cell lysis is an inevitable step in classical affinity purification - mass spectrometry (AP-MS) strategies to map intracellular protein interactions. Cell homogenization implies a drastic change of the protein complex environment which strongly impedes comprehensive analysis. Complementary lysis conditions, in situ crosslinking strategies and proximal labeling techniques have been recently developed to address part of this problem. We here demonstrate the use of a viral particle sorting approach as an alternative lysis-free method. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners of the bait protein become trapped within virus-like particles (VLPs) that bud from mammalian cells. This so-called Virotrap approach obviates the need for cell homogenization and preserves the protein complexes during purification. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions as well as MS-based identification of novel protein partners. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the current arsenal of MS-based methods to study protein complexes.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Sven Eyckerman

PROVIDER: MSV000080790 | MassIVE | Wed Mar 29 15:18:00 BST 2017

SECONDARY ACCESSION(S): PXD000685

REPOSITORIES: MassIVE

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Virotrap: Abducting Protein Complexes from Mammalian Cells

Project description:Cell lysis is an inevitable step in classical affinity purification - mass spectrometry (AP-MS) strategies to map intracellular protein interactions. Cell homogenization implies a drastic change of the protein complex environment which strongly impedes comprehensive analysis. Complementary lysis conditions, in situ crosslinking strategies and proximal labeling techniques have been recently developed to address part of this problem. We here demonstrate the use of a viral particle sorting approach as an alternative lysis-free method. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners of the bait protein become trapped within virus-like particles (VLPs) that bud from mammalian cells. This so-called Virotrap approach obviates the need for cell homogenization and preserves the protein complexes during purification. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions as well as MS-based identification of novel protein partners. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the current arsenal of MS-based methods to study protein complexes.

2016-04-27 | PXD000685 | Pride

Controlling for gene expression changes in transcription factor protein networks.

Project description:The development of affinity purification technologies together with mass spectrometric analyses of the purified protein mixtures (AP-MS) has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we have investigated whether ectopic expression of an affinity tagged transcription factor as bait in AP-MS experiments perturbs gene expression in cells resulting in false positive identification of bait associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA-Seq, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then copurify non-specifically and be misidentified as bait associated proteins. Therefore typical controls should be sufficient and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFêB1, NFêB2, Rel, RelB, IêBá, IêBâ and IêBå). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFêB family transcription factors. The work here therefore provides a conceptual and experimental framework for analyzing transcription faction protein interactions. Gene expression profiles were assayed in triplicate from HEK293 cells expressing either Halo-RelA, Halo-NFkB1, or Halo tag alone.

2014-04-24 | E-GEOD-57027 | biostudies-arrayexpress

Controlling for gene expression changes in transcription factor protein networks.

2014-04-24 | GSE57027 | GEO

Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors (Sap30bp RNA-Seq)

Project description:Alternative splicing is fundamental for the expansion of biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental stage and tissue-dependent alternative exons often control protein-protein interactions, yet only a minor fraction of these events has been characterized at the protein level. Using affinity purification-mass spectrometry (AP-MS) we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners. Focusing on Chtop and Sap30bp, the neural exons in these proteins unexpectedly remodel interactions with partners associated with mRNA processing and trafficking. Sap30bp additionally controls RNA localization by regulating the splicing of <100 nt ‘mini-introns’ that contribute to the nuclear retention of transcripts. These activities of Chtop and Sap30bp are linked to programs of transcriptomic regulation associated with development of the nervous system. AP-MS is thus a powerful approach for elucidating multifaceted functions of proteins imparted by context-dependent alternative exons.

2023-11-24 | GSE224970 | GEO

Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors (Chtop RNA-Seq)

2023-11-24 | GSE224969 | GEO

Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors (iCLIP-Seq)

2023-11-24 | GSE224968 | GEO

In vivo protein-protein interaction in C. elegans early embryos

Project description:We combine in vivo expressed GFP-tagged proteins with quantitative proteomics to identify protein-protein interactions of selected key proteins involved in early C. elegans embryogenesis. Co-affinity purification of interaction partners for eight bait proteins resulted in a pilot in vivo interaction map of proteins with a focus on early development.

2016-02-29 | PXD002624 | Pride

Identification of binding proteins for PARP1 by TurboID

Project description:To identify novel interaction partners of PARP1, we engineered a fusion of the full-length PARP1 with the TurboID enzyme and two negative controls. The first control omitted the bait protein PARP1, excluding the potential interaction of the TurboID protein itself. However, it bears the drawback of potentially obscuring real interactions due to the high flexibility and activity of free TurboID. The second control omitted exogenous biotin, serving as a complementary measure to address this concern.

2024-02-01 | PXD049100 |

protein-protein interaction dataset TNFR1-mediated NFkB signaling

Project description:Most MS-based approaches to study protein-protein interactions require homogenization of the cells, which leads to loss of the cellular structures, dilution of proteins and formation of incorrect protein complexes. Furthermore, several purification and washing steps are needed, which can lead to the loss of (weaker) interactions. We recently developed a new method for studying protein complexes without interfering with the cellular integrity; Virotrap. By trapping and purifying protein complexes in virus-like particles, we avoid cell lysis and preserve intact complexes during the purification process. Here, we present a protein-protein interaction network of the TNFR1-induced NF-κB and apoptosis pathway, using TRADD, FADD, TRAF2, cIAP1, NEMO, TANK, HOIL1, HOIP, SHARPIN, A20, ABIN1 and ABIN2 as baits in Virotrap. Virotrap was applied both under resting conditions as well as upon TNF stimulation, which reveals dynamic interactions in TNFR1-induced NF-κB signaling. The found interactions compromise both known interactors as well as novel ones that might serve as valuable resource for further unraveling the molecular mechanisms controlling inflammation, cellular proliferation and cell death.

2018-11-06 | PXD009366 | Pride

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions

Project description:Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein “bait”. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs wes analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.

2018-10-20 | GSE121524 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data