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PXD016628

PXD016628 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleMudPIT analysis of STRN4 interacting proteins from HEK TER cells expressing either SV40 ST or GFP
DescriptionHEK TER cells expressing either SV40 ST or GFP (30 x 15-cm diameter plates) were harvested with lysis buffer (20 mM imidazole HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0 with 10 ug/mL each of aprotinin, leupeptin, pepstatin, 1 mM benzamidine, and 1 mM PMSF). The clarified cell extract was incubated overnight at 4C with 20-100 ug of STRN4 antibodies (Abcam, ab177155) crosslinked to 30 mg protein A agarose beads (Thermo Scientific) by dimethyl pimelimidate (DMP). Beads were washed 5 times with high salt lysis buffer (containing 300 mM NaCl), washed with TBS 2 times, and then eluted with 0.2 M glycine pH 3 and neutralized with 1 M Tris-HCl pH 8.0. Proteins were precipitated with trichloroacetic acid (20% final concentration) overnight at 4C, washed with cold acetone and processed for subsequent MudPIT analysis. In brief, TCA-precipitated protein eluates were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. The peptide mixtures were loaded onto microcapillary fused silica columns (100um i.d.), packed with C18 reverse phase (Aqua; Phenomenex), SCX (Luna; Phenomenex) and C18-RP, placed in-line with an Agilent 11000 quaternary pump, and analyzed by a 10-step MudPIT on linear ion traps. MS/MS datasets were searched using ProLuCID against a non-redundant human protein database (NCBI, 2019-12-03) containing 44,080 non-redundant human proteins, 426 usual contaminants, as well as the sequences for small and large T antigens from SV40 Macaca mulatta polyomavirus 1. To estimate false discover rates (FDRs), the amino acid sequence of each non-redundant protein was randomized (44,521 shuffled proteins) and added to the search space. Cysteine carboxylation was searched as a static modification while methionine oxidation was searched dynamically. Peptide/spectrum matches were sorted and selected using DTASelect in combination with an in-house software, swallow, to FDRs at the peptide and protein levels of less than 1%.
HostingRepositoryMassIVE
AnnounceDate2019-12-05
AnnouncementXMLSubmission_2019-12-05_10:35:29.xml
DigitalObjectIdentifier
ReviewLevelNon peer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportSupported dataset by repository
PrimarySubmitterLaurence Florens
SpeciesList scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606;
ModificationListS-carboxamidomethyl-L-cysteine; L-methionine sulfoxide
InstrumentLTQ
Dataset History
RevisionDatetimeStatusChangeLog Entry
02019-12-05 10:28:17ID requested
12019-12-05 10:35:30announced
Publication List
no publication
Keyword List
submitter keyword: MAP4K4, PP2A-mediated dephosphorylation, Striatin-interacting phosphatase and kinase (STRIPAK) complex, SV40 Small T Antigen
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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