MassIVE MSV000082907

Partial Public PXD011016

MudPIT analyses of the proteins co-purified with the junction-localized tumor suppressors Scribble (Scrib) and Discs Large (Dlg) by immunoprecipitation from fly embryos

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Description

Fly embryos (w1118, Dlg-GFP, or Scrib-GFP) were lysed with lysis buffer (25mM Tris-HCl, 150mM EDTA, 1% NP-40, 5% glycerol, 1mM DTT, 1x protease inhibitor). Debris was removed by centrifugation, and extracts were cleared by incubation with Dynabeads (1 hour). Immunocomplexes were formed by incubation for 2 hours with Dlg antibody-conjugated magnetic beads or GFP-nanobody (GFP-Trap_MA, ChromoTek). Immunocomplexes were washed with wash buffer (25mM Tris-HCl, 150mM EDTA), eluted with elution buffer (50mM glycine PH 2.5, 150mM NaCl), and then neutralized with Tris-HCl PH 9.5. Two biological replicates of Dlg-IPs (and their corresponding negative controls without antibody) and two GFP-IPs from flies expressing either Dlg-GFP or Scrib-GFP (and their negative GFP-IP control) were analyzed by MudPIT mass spectrometry. 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.), 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 SEQUEST (Eng et al., 1994) against a D. melanogaster database (NCBI, 2012-03-08 release) containing 18,564 non-redundant proteins and 177 usual contaminants (human keratins, IgGs, and proteolytic enzymes). To estimate false discover rates (FDRs), the amino acid sequence of each non-redundant protein was randomized and searched at the same time. Peptide/spectrum matches were sorted and selected using DTASelect (Tabb et al., 2002) with the following criteria set: spectra/peptide matches were retained only if they had a DeltCn of at least 0.8, and minimum XCorr of 1.8 for singly, 2.5 for doubly, and 3.5 for triply charged spectra. Additionally, the peptides had to be minimum 7 amino acids in length and fully tryptic. The FDRs were 0.5 +/- 0.3 and 1.3 +/- 0.8 at the peptide and protein levels, respectively. Peptide hits from multiple runs were compared using CONTRAST (Tabb et al., 2002). The distributed normalized spectral abundance factors (dNSAF) were used to estimate relative protein levels (Zhang et al., 2010). [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Discs Large (Dlg) ; Scribble (Scrib) ; planar mitotic spindle orientation

Contact

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

Publications

Nakajima YI, Lee ZT, McKinney SA, Swanson SK, Florens L, Gibson MC.
Junctional tumor suppressors interact with 14-3-3 proteins to control planar spindle alignment.
J. Cell Biol. 2019 Jun 3;218(6):1824-1838. Epub 2019 May 14.

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