MassIVE MSV000083305

Complete Public PXD012273

MudPIT analyses of the proteins co-purified with wild type-Hzg-3XHA/Flag and phosphatase dead-Hzg-3XHA/Flag affinity purifications from Drosophila melanogaster S2 cells

Description

Protein Complexes Purification- Stable S2 cell expressing Hzg albeit low level, lines expressing C-terminally 3XHA-Flag-tagged wild-type and phosphatase dead (PD) Hzg were generated. A parental cell line was used as a negative control. Nuclear and cytoplasmic extracts from the Hzg-WT, Hzg-mutant and Control cell lines were generated. The Hzg protein complex was immunopurified using anti-Flag antibodies. Three biological replicates were generated for each sample type. Bead bound proteins were identified using Multidimensional Protein Identification technology (MudPIT). 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. 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 SEQUEST against a database consisting of 21,402 non-redundant Drosophila melanogaster proteins (downloaded from NCI RefSeq 2013-02-20), 1 phosphatase dead Hzg sequence, 177 usual contaminants, and, to estimate false discovery rates (FDRs), 21,579 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 and compared using DTASelect/Contrast. [doi:10.25345/C5Q896] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Herzog, Hzg ; prion-like protein ; phosphatase ; segment polarity ; amyloid

Contact

Principal Investigators:
(in alphabetical order)
Anita Saraf, The Stowers Institute for Medical Research, United States
Submitting User: asaraf

Publications

Nil Z, Hervás R, Gerbich T, Leal P, Yu Z, Saraf A, Sardiu M, Lange JJ, Yi K, Unruh J, Slaughter B, Si K.
Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.
Cell. 2019 Sep 5;178(6):1403-1420.e21.

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