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PXD010462

PXD010462 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleMudPIT analyses of the proteins co-purified with FLAG-HA Non-Stop FLAG-IPed from Drosophila melanogaster S2 cells
DescriptionProtein 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.
HostingRepositoryMassIVE
AnnounceDate2019-08-01
AnnouncementXMLSubmission_2019-08-01_03:04:27.xml
DigitalObjectIdentifier
ReviewLevelNon peer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterLaurence Florens
SpeciesList scientific name: Drosophila melanogaster; common name: fruit fly; NCBI TaxID: 7227;
ModificationListL-methionine sulfoxide; unknown modification: [C] [57.021464] S-carboxamidomethyl-L-cysteine
InstrumentLTQ; LTQ Orbitrap
Dataset History
RevisionDatetimeStatusChangeLog Entry
02018-07-17 11:45:20ID requested
12019-08-01 03:05:00announced
12019-08-01 03:05:00announced
12019-08-01 03:05:00announced
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 affiliationThe Stowers Institute for Medical Research
contact emaillaf@stowers.org
lab head
Laurence Florens
contact affiliationStowers Institute for Medical Research
contact emaillaf@stowers.org
dataset submitter
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