Dataset Information

MudPIT analyses of the proteins co-purified with FLAG-HA Non-Stop FLAG-IPed from Drosophila melanogaster S2 cells

ABSTRACT: Protein Complexes Purification- Drosophila S2 cells were stably-transfected with pRmHa3-based inducible expression vectors to produce epitope-tagged Non-stop-2xFLAG-2xHA (Non-stop-FH). Cells stably transfected with empty vector served as negative control. Protein complexes containing Non-stop were purified from nuclear extracts via their FLAG epitope tag. Isolated complexes were further separated by size using Superose 6 gel filtration chromatography. Eluates from the FLAG-IP and SEC purifications were TCA-precipitated from proteomics analysis. Three biological replicates of the SEC group 2 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. The digested Not and control FLAG-IP eluates were analyzed on an LTQ-Orbitrap (Thermo) coupled to an Eksigent NanoLC-2D. In both cases, 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 the long (703 amino acids) isoform of non-stop, 22,006 non-redundant Drosophila melanogaster proteins (merged and deduplicated entries from GenBank release 6, FlyBase release 6.2,2 and NCI RefSeq release 88), 225 usual contaminants, and, to estimate false discovery rates (FDRs), 22,007 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 Orbitrap, LTQ

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

SUBMITTER: Laurence Florens

PROVIDER: MSV000082625 | MassIVE | Tue Jul 17 11:45:00 BST 2018

SECONDARY ACCESSION(S): PXD010462

REPOSITORIES: MassIVE

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Publications

Ataxin-7 and Non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization.

Cloud Veronica V Thapa Ada A Morales-Sosa Pedro P Miller Tayla M TM Miller Sara A SA Holsapple Daniel D Gerhart Paige M PM Momtahan Elaheh E Jack Jarrid L JL Leiva Edgardo E Rapp Sarah R SR Shelton Lauren G LG Pierce Richard A RA Martin-Brown Skylar S Florens Laurence L Washburn Michael P MP Mohan Ryan D RD

eLife 20190726

Atxn7, a subunit of SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the Atxn7 amino terminus anchors Non-stop, a deubiquitinase, to the complex. To understand the scope of Atxn7-dependent regulation of Non-stop, substrates of the deubiquitinase were sought. This revealed Non-stop, dissociated from Atxn7, interacts with Arp2/3 and WAVE reg ...[more]

PMID: 31348003

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