MassIVE MSV000084662

Complete Public PXD016628

MudPIT analysis of STRN4 interacting proteins from HEK TER cells expressing either SV40 ST or GFP

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Description

HEK 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%. [doi:10.25345/C5QX1Q] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: MAP4K4 ; PP2A-mediated dephosphorylation ; Striatin-interacting phosphatase and kinase (STRIPAK) complex ; SV40 Small T Antigen

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens

Publications

Kim JW, Berrios C, Kim M, Schade AE, Adelmant G, Yeerna H, Damato E, Iniguez AB, Florens L, Washburn MP, Stegmaier K, Gray NS, Tamayo P, Gjoerup O, Marto JA, DeCaprio J, Hahn WC.
STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells.
Elife. Epub 2020 Jan 8. pii: e53003. doi: 10.7554/eLife.53003.

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