Protein Complexes Purification- Stable S2 cell lines expressing Ada2b (isoform B; NP_001027151.1) in the pRmHa3-CHA2FL2 (Ada2b-PBH2F2) were maintained at 1-2 e6 cells/mL in SFX medium (HyQ SFX-Insect). Cells stably transfected with empty vector served as negative controls. Protein complexes containing Ada2b-PB were purified from nuclear extracts (from 12 L of 1e7/L S2 Ada2b-PBH2F2 cells) via their FLAG epitope tag. Isolated complexes were further separated by size on a Superose 6 10/300 gel filtration column. Eluates from the FLAG-IP (input) and size exclusion chromatography (SEC) fractions were TCA-precipitated from proteomics analysis. Two biological replicates of SEC fractions 12 and 19 were analyzed.
Multidimensional Protein Identification Technology- TCA-precipitated protein pellets were solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Roche) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The digested size exclusion eluates were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100.
MS Data Processing- The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 21,402 non-redundant Drosophila melanogaster proteins (downloaded from NCI RefSeq 2013-02-20), 177 usual contaminants, and, to estimate false discovery rates (FDRs), 21,579 randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues. To account for the oxidation of methionine to methionine sulfoxide, 16 Da were added as a differential modification to the methionine residue. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: acetyl transferase ; SAGA complex ; ADA complex
|Principal Investigators:||Laurence Florens, The Stowers Institute for Medical Research, USA|
Soffers JHM, Li X, Saraf A, Seidel CW, Florens L, Washburn MP, Abmayr SM, Workman JL.
Characterization of a metazoan ADA acetyltransferase complex.
Nucleic Acids Res. 2019 Apr 23;47(7):3383-3394.