Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

System-wide profiling of protein-complexes via size exclusion chromatography– mass spectrometry (SEC-MS)

ABSTRACT: In living cells most proteins are organized in stable or transient functional assemblies known as protein complexes, which control a multitude of vital cellular processes such as cell cycle progression, metabolism, and signal transduction. System-wide workflows for analysis of protein complexes using mass spectrometry based proteomics consists of a biochemical fractionation methods which take advantage of biophysical features such as charge or mass of protein complexes. In this project, we apply size exclusion chromatography to investigate protein assemblies from a Jurkat whole cell lysate. We introduced a fast and robust in-plate sample preparation workflow which allows for replicates to be performed with fewer technical hurdles and with a high degree of reproducibility) We acquired the data in SWATH-mode in combination with a high-throughput LC-system (EVOSEP one). The bioinformatics pipeline builds on the OpenSWATH workflow combined with CCprofiler, a tool for statistical scoring of protein co-elution profiles. This method enabled us to identify more than 100 protein complexes from mammalian cells within one day of measurement.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Jurkat Cell

SUBMITTER: Fabian Frommelt

LAB HEAD: , Ruedi Aebersold

PROVIDER: PXD018033 | Pride | 2022-05-06

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	2020_Methods_in_molecular_biology_information_file.txt	Txt
	HS_decoy_EvosepLib.pqp	Other
	combined_iRT_CiRT_201804.TraML	Other
	feature_alignment.csv	Csv
	vmatej_I190208_001.mzXML.gz	Mzxml

Items per page:

1 - 5 of 187

Similar Datasets

ChIP-seq of H2A.Z and its histone chaperones during the cell cycle

Project description:The aim of this study was to uncover cell cycle dependent chromatin binding of H2A.Z and its histone chaperones. U2OS cells were stably transfected with Twin-Strep-tagged H2A.Z, ANP32e, or YL1 constructs, and WT cells were used as a negative/background control. U2OS cells were synchronised in G1 or G2-M phase using hydroxyurea or nocodazole, respectively, and protein-DNA complexes were isolated by affinity purification.

2023-06-29 | E-MTAB-11617 | biostudies-arrayexpress

Rapid profiling of protein complex re-organization in perturbed systems

Project description:Protein complexes constitute the primary functional modules of cellular activity. To respond to perturbations, complexes undergo changes in their abundance, subunit composition or state of modification. Understanding the function of biological systems requires global strategies to capture this contextual state information on protein complexes and interaction networks. Methods based on co-fractionation paired with mass spectrometry have demonstrated the capability for deep biological insight but the scope of studies using this approach has been limited by the large measurement time per biological sample and challenges with data analysis. As such, there has been little uptake of this strategy beyond a few expert labs into the broader life science community despite rich biological information content. We present a rapid integrated experimental and computational workflow to assess the re-organization of protein complexes across multiple cellular states. It enables complex experimental designs requiring increased sample/condition numbers. The workflow combines short gradient chromatography and DIA/SWATH mass spectrometry with a data analysis toolset to quantify changes in complex organization. We applied the workflow to study the global protein complex rearrangements of THP-1 cells undergoing monocyte to macrophage differentiation and a subsequent stimulation of macrophage cells with lipopolysaccharide. We observed massive proteome organization in functions related to signaling, cell adhesion, and extracellular matrix during differentiation, and less pronounced changes in processes related to innate immune response induced by the macrophage stimulation. We therefore establish our integrated differential pipeline for rapid and state-specific profiling of protein complex organization with broad utility in complex experimental designs.

2023-06-27 | PXD036711 | Pride

Transcription profiling of yeast deletion mutants of putative protein complex members

Project description:A number of putative protein complexes have been selected for experimental validation by gene expression profiling of the individual deletion mutants of the respective protein complex members.

2010-06-01 | E-TABM-654 | biostudies-arrayexpress

psiCLIP of SmB, Prp16 and Prp22 in C, C* and P spliceosomal complexes

Project description:Despite the major progress made into spliceosome mechanisms through CryoEM, a major obstacle of this approach is its inability to map the positioning of helicases on the RNA substrate. Here we present a method ‘psiCLIP’ which probes protein-RNA interactions in specific spliceosomal states. The method is based on iCLIP, but is different to previous iCLIP studies in that it is performed on step-specific spliceosomes that are prepared from in vitro splicing extracts, similarly to those prepared for CryoEM. We applied psiCLIP to SmB (C complex), Prp16 (C complex) and Prp22 (C* and P complexes), using pre-mRNA substrates based on UBC4 and ACT1. We also used ATPase deficient dominant negative (dn) mutants of Prp16 and Prp22 to determine the contribution of ATPase activity to binding patterns.

2020-03-15 | E-MTAB-8895 | biostudies-arrayexpress

Extensive characterization of NF-kappaB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits

Project description:We report the DNA binding preferences of 4 NF-kappaB dimers (p52p52, RELARELA, RELAp50, RELAp52) Proteins of the Nf-kappab family were bound to DNA oligonucleotides containing a degenerate region. The protein-DNA complexes were selected after one or multiple rounds of SELEX and the DNA molecules were deep sequenced.

2011-05-24 | E-GEOD-29460 | biostudies-arrayexpress

D2O proteome dynamics in FSHD vs UASb myoblasts

Project description:Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight to disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labelling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from 2 individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4483 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of ‘older’ less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

2023-06-27 | PXD038818 | Pride

A non-canonical SWI/SNF complex underlies synthetic lethality in cancers driven by BAF complex perturbation [RNA-seq]

Project description:Mammalian SWI/SNF chromatin remodeling complexes exist in three distinct, final-form assemblies: canonical BAF (cBAF), PBAF, and a newly characterized non-canonical complex, ncBAF. However, their complex-specific targeting on chromatin, functions and roles in disease remain largely unknown. Here, we comprehensively map complex assemblies on chromatin and find that ncBAF uniquely localizes to CTCF sites and promoters. We identified ncBAF subunits as major synthetic lethalities specific to human synovial sarcoma and malignant rhabdoid tumor, which share in common cBAF complex perturbation. Chemical degradation of the BRD9 subunit of ncBAF rapidly attenuates SS and MRT cell proliferation. Notably, in cBAF-perturbed cancers, ncBAF complexes retain their hallmark localization to CTCF sites and promoters, and maintain gene expression at retained mSWI/SNF sites to support cell proliferation in a manner distinct from fusion oncoprotein-mediated targeting. Taken together, these findings unmask the unique targeting and function of ncBAF complexes and present new cancer-specific therapeutic targets.

2018-11-04 | GSE113041 | GEO

A non-canonical SWI/SNF complex underlies synthetic lethality in cancers driven by BAF complex perturbation [ChIP-seq]

2018-11-04 | GSE113040 | GEO

ACD15, ACD21 and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements [ChIP-seq]

Project description:DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG-binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here we characterize two additional complex members: α-crystalline domain containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of complex components regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of gene silencing complexes that act to drive the formation of higher order, dynamic assemblies.

2023-09-14 | GSE234531 | GEO

ACD15, ACD21 and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements [RNA-seq]

2023-09-14 | GSE234530 | GEO