Dataset Information

AP-MS analysis of human histone deacetylase interactions in CEM-T cells

ABSTRACT: Affinity purification-mass spectrometry (AP-MS) datasets of eleven human histone deacetylases (HDACs). Affinity-tagged (GFP) HDACs were expressed in a human CEM-T cell line. For label-free analysis, proteins were digested by trypsin in-solution and peptides separated across a linear 180 min gradient by a one-dimensional LC-MS/MS analysis. For label-free analysis, MS data (RAW files) were converted into mzXML format using the Proteowizard conversion tool. mzXML files were searched using the X!Tandem/k-score database search tool against the human subset of the UniProt protein sequence database, appended with a list of common sample contaminants. The search results were processed using PeptideProphet and ProteinProphet tools. The data from individual experiments across 7 biological replicates of GFP controls and 23 HDAC-GFP-tagged biological replicates were merged, and the spectral counts were extracted using the software, ABACUS. The combined list of protein identifications (protein groups) was filtered to achieve a protein-level FDR of less than 1%. When computing the spectral counts in individual experiments, proteins were quantified (i.e. spectra counted) if they were identified with a probability equal or greater than 0.9 in that particular replicate. For metabolic labeling IDIRT experiments, isolated proteins were separated by SDS-PAGE, digested by trypsin in-gel, and peptides were separated across a linear 90 min gradient. RAW files were processed and peptides were identified (less than 1% FDR) and quantified using SEQUEST/Percolator in the Proteome Discoverer software (v1.3). IDIRT protein ratios were normalized by the median SILAC protein ratio determined from 1D-LC-MS/MS analysis of input lysates. Protein interactions have been submitted to the IMEx consortium through IntAct with the identifier IM-18733.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Todd Greco

PROVIDER: PXD000208 | Pride | 2013-06-14

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2011-01-20_HDAC1_GFP_01.mzXML.gz	Mzxml
	2011-01-20_HDAC1_GFP_01.raw	Raw
	2011-01-20_HDAC1_GFP_02.mzXML.gz	Mzxml
	2011-01-20_HDAC1_GFP_02.raw	Raw
	2011-02-08_HDAC3_GFP_03.mzXML.gz	Mzxml

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Publications

The functional interactome landscape of the human histone deacetylase family.

Joshi Preeti P Greco Todd M TM Guise Amanda J AJ Luo Yang Y Yu Fang F Nesvizhskii Alexey I AI Cristea Ileana M IM

Molecular systems biology 20130101

Histone deacetylases (HDACs) are a diverse family of essential transcriptional regulatory enzymes, that function through the spatial and temporal recruitment of protein complexes. As the composition and regulation of HDAC complexes are only partially characterized, we built the first global protein interaction network for all 11 human HDACs in T cells. Integrating fluorescence microscopy, immunoaffinity purifications, quantitative mass spectrometry, and bioinformatics, we identified over 200 unr ...[more]

PMID: 23752268

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