PXD010462
PXD010462 is an original dataset announced via ProteomeXchange.
Dataset Summary
| Title | MudPIT analyses of the proteins co-purified with FLAG-HA Non-Stop FLAG-IPed from Drosophila melanogaster S2 cells |
| Description | 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. |
| HostingRepository | MassIVE |
| AnnounceDate | 2019-08-01 |
| AnnouncementXML | Submission_2019-08-01_03:04:27.xml |
| DigitalObjectIdentifier | |
| ReviewLevel | Non peer-reviewed dataset |
| DatasetOrigin | Original dataset |
| RepositorySupport | Unsupported dataset by repository |
| PrimarySubmitter | Laurence Florens |
| SpeciesList | scientific name: Drosophila melanogaster; common name: fruit fly; NCBI TaxID: 7227; |
| ModificationList | L-methionine sulfoxide; unknown modification: [C] [57.021464] S-carboxamidomethyl-L-cysteine |
| Instrument | LTQ; LTQ Orbitrap |
Dataset History
| Revision | Datetime | Status | ChangeLog Entry |
|---|---|---|---|
| 0 | 2018-07-17 11:45:20 | ID requested | |
| ⏵ 1 | 2019-08-01 03:05:00 | announced | |
| ⏵ 1 | 2019-08-01 03:05:00 | announced | |
| ⏵ 1 | 2019-08-01 03:05:00 | announced |
Publication List
| no publication |
Keyword List
| submitter keyword: Non-stop deubiquitinase, spinocerebellar ataxia type 7, SAGA complex, Arp2/3 complex, WAVE complex |
Contact List
| Laurence Florens | |
|---|---|
| contact affiliation | The Stowers Institute for Medical Research |
| contact email | laf@stowers.org |
| lab head | |
| Laurence Florens | |
| contact affiliation | Stowers Institute for Medical Research |
| contact email | laf@stowers.org |
| dataset submitter | |
Full Dataset Link List
| MassIVE dataset URI |
| Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://massive.ucsd.edu/MSV000082625/ |
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