Proteomics

Dataset Information

0

MudPIT analyses of the proteins co-purified with Ada2b-PB-FLAG-HA FLAG-affinity purified from Drosophila melanogaster S2 cells


ABSTRACT: 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.

INSTRUMENT(S): LTQ

ORGANISM(S): Drosophila Melanogaster (ncbitaxon:7227)

SUBMITTER: Laurence Florens  

PROVIDER: MSV000083153 | MassIVE | Tue Nov 20 13:55:00 GMT 2018

SECONDARY ACCESSION(S): PXD011770

REPOSITORIES: MassIVE

altmetric image

Publications

Characterization of a metazoan ADA acetyltransferase complex.

Soffers Jelly H M JHM   Li Xuanying X   Saraf Anita A   Seidel Christopher W CW   Florens Laurence L   Washburn Michael P MP   Abmayr Susan M SM   Workman Jerry L JL  

Nucleic acids research 20190401 7


The Gcn5 acetyltransferase functions in multiple acetyltransferase complexes in yeast and metazoans. Yeast Gcn5 is part of the large SAGA (Spt-Ada-Gcn5 acetyltransferase) complex and a smaller ADA acetyltransferase complex. In flies and mammals, Gcn5 (and its homolog pCAF) is part of various versions of the SAGA complex and another large acetyltransferase complex, ATAC (Ada2A containing acetyltransferase complex). However, a complex analogous to the small ADA complex in yeast has never been desc  ...[more]

Similar Datasets

2018-07-17 | MSV000082625 | MassIVE
2019-01-08 | MSV000083305 | MassIVE
2013-10-31 | PXD000553 | Pride
2020-03-20 | MSV000085126 | MassIVE
2019-02-20 | MSV000083465 | MassIVE
2019-12-10 | MSV000084671 | MassIVE
2018-06-28 | MSV000082521 | MassIVE
2019-11-13 | MSV000084579 | MassIVE
2020-03-20 | MSV000085125 | MassIVE
2022-10-25 | PXD026639 | Pride