CSHL_EIF4A2_FLAGAP_2022_P36_V35
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Anne-Claude Gingras
PROVIDER: MSV000092534 | MassIVE | Thu Jul 27 05:04:00 BST 2023
REPOSITORIES: MassIVE
Similar Datasets
Project description:Test set for CSHL Proteomics Course
Contains set of 8 samples acquired on an Orbitrap Fusion Lumos Tribrid.
Searched with Fragpipe v19.1.
2023-07-27 | MSV000092533 | MassIVE
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
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.
2014-04-24 | GSE57027 | GEO
Project description:Affinity purification coupled with mass spectrometry (AP-MS) and proximity-dependent biotinylation identification (BioID) methods are powerful tools to define the interactome for a specific protein bait. Whereas AP-MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. In order to comprehensively characterize the IBTK interactome networks in cells, we developed a tag workflow which allows for both AP-MS and BioID analysis with a single construct, pcDNA5/FRT vector containing FLAG-BirA-IBTK.
2024-05-10 | PXD038286 | Pride
Project description:In this study we described the protein-protein interaction network of the Drosophila Speciation Core Complex by analysing the interactome of its subunit: HMR, LHR, NLP, BOH1 (CG33213), BOH2 (CG4788) and HP1a. For this purpose we performed Affinity Purification coupled with Mass Spectrometry (AP-MS) in D. melanogaster SL2 cells using as bait the two hybrid incompatibility proteins HMR (n = 8) and LHR (n = 4), as well as NLP (n = 3), BOH1(CG33213, n = 4), BOH2 (CG4788, n = 5) and HP1a (n = 4). Each bait was targeted with at least one antibody (rat anti-LHR 12F4, mouse anti-HP1a 2C09, rabbit anti-Nlp, anti-FLAG-M2 for FLAG-CG33213 and FLAG-CG4788), while HMR was targeted with three different antibodies (rat anti-HMR 2C10 and 12F1, anti-FLAG-M2 for FLAG-HMR). Individual replicates and antibodies used are listed in samples_table.
2021-08-11 | PXD023188 | Pride
Project description:The quantitative multiplexing capacity of isobaric Tandem Mass Tags (TMT) has increased the throughput of affinity purification mass spectrometry (AP-MS) to characterize protein interaction networks of immunoprecipitated baits. However, variable bait levels between replicates can convolute interactor identification. We compared the Student's t difference test and Pearson's R correlation as methods to generate t-statistics and assessed the significance of interactors following TMT-AP-MS. Using a simple linear model of protein recovery in immunoprecipitates to simulate reporter ion ratio distributions, we found that correlation-derived t-statistics protect against bait variance while robustly controlling Type I errors (false positives). We experimentally determined the performance of these two approaches for determining t-statistics under two experimental conditions: irreversible prey association to the Hsp40 mutant DNAJB8H31Q followed by stringent washing, and reversible association to 14 3 3 with gentle washing. Correlation-derived t-statistics performed at least as well as difference test t-statistics for each sample, with substantial improvement in performance for experiments with high bait level variance. Deliberately varying bait levels over a large range fails to improve selectivity but does increase robustness between runs. The use of correlation-derived t-statistics should improve identification of interactors using TMT-AP-MS.
2020-06-22 | PXD016613 | Pride
Project description:There are several methods to bring down the bait and interactors from the cell lysates, each with its own advantages and disadvantages. Recently, the most commonly used method is one step AP, TAP and proximity-labeling. In order to analyze the Strengths and weaknesses of the three methods, fusion proteins were constructed by NICD4 with FLAG, SFB (S tag-2x Flag tag-SBP tag) and TurboID respectively, NICD4 and its interactors were obtained by purification. By analyzing these proteins, we believe that SFB-TAP is the most reliable and effective method to identify interacting proteins.
2022-08-12 | PXD031772 | Pride
Project description:To identify the proteins interact with PDL1 protein in human cell lines, we analyzed the protein complexes using tandem affinity purification followed by mass spectrometry (TAP-MS) in HEK293T. We established the cell lines stably expressing PDL1 gene fused with Flag tags. We identified proteins associated with the bait in the isolated complexes using MS and searched the human databases for these proteins.
2022-10-13 | PXD031702 | Pride
Project description:We report the identification of new noncoding RNAs in Brucella suis 1330 and that are associated to the chaperone protein Hfq Coimmunoprecipitation using Flag-tagged Hfq as bait
2015-11-25 | E-GEOD-73621 | biostudies-arrayexpress
Project description:PUP-IT proximity labeling of Arabidopsis Target of Rapamycin Complex (TORC), using the yeast FKBP protein (P20081) as a bait with and without rapamycin treatment and constitutive expression of FLAG::PUP(E). The sample metadata, sequence database and fragpipe workflow are uploaded under the 'Metadata' tag.
2025-01-16 | MSV000096864 | MassIVE