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):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Todd Greco  

LAB HEAD: Todd Greco

PROVIDER: PXD000208 | Pride | 2013-06-14

REPOSITORIES: Pride