Project PXD000172

Summary

Title

Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes

Description

Methylation of lysine 4 at histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multi-subunit (SET1/MLL) complexes have been described that contain methyltransferase activity towards H3K4, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. Peptides were applied to online nanoLC-MS/MS, using a 120 min acetonitrile gradient (5.6 - 76%). Mass spectra were recorded on an LTQ-Orbitrap-Velos mass spectrometer (Thermo) selecting the 15 most intense precursor ions of every full scan for fragmentation. Raw data were analyzed using MaxQuant 1.3.0.5, with label-free quantification (LFQ), match between runs (between triplicates) and the iBAQ algorithm enabled. MaxQuant default settings were used for peptide identification; Enzyme: Trypsin/P, MS tolerance (FTMS): 6 ppm, max missed cleavages: 2, max charge: 7, MS/MS tolerance (ITMS): 0.5 Da, Peptide FDR: 0.01 and Protein FDR: 0.01. Normalized mass spectrometric intensities (LFQ intensities) were compared between the GFP-tagged and control sample, using an adapted permutation-based false discovery rate (FDR) t-test in Perseus (MaxQuant package).

Sample Processing Protocol

See details in reference PMID : 23508102

Data Processing Protocol

See details in reference PMID : 23508102

Contact

A.H. Smits, University Medical Center Utrecht

Submission Date

04/03/2013

Publication Date

13/09/2013

Tissue

Not available

Instrument

LTQ Orbitrap Velos

Software

Not available

Experiment Type

Bottom-up proteomics

Publication

    van Nuland R, Smits AH, Pallaki P, Jansen PW, Vermeulen M, Timmers HT; Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes., Mol Cell Biol, 2013 May, 33, 10, 2067-77, PubMed(s) : 23508102