MassIVE MSV000085815

Complete Public PXD020533

MudPIT analyses of FLAG-DYRK1A and FLAG-Controls affinity-purified from cytoplasmic extracts of HEK293 cells

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Description

Approximately 10E9 cells for each cell line (FLAG-DYRK1A, FLAG-DYRK1A-K188R, and FLAG-controls) were collected and washed with PBS. Cells were swollen for 15 minutes in hypertonic buffer (Buffer A: 10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) freshly supplemented with protease inhibitor cocktail (Sigma P8340). Swollen cells were dounced 20 times in a Wheaton dounce homogenizer till about 90% cells were lysed (as observed in a microscope). Lysate is then centrifuged at 25Kg for 20 min, to pellet Nuclei and cell debris. To the supernatant (S100) 0.11 volume of Buffer B (0.3 M HEPES pH 7.9, 1.4 M KCl and 0.03 M MgCl2) was added and cleared by centrifuging at 100Kg, 4C for 1 hour. The supernatant was treated as cytoplasmic extract, and Flag-affinity was performed by incubating Flag beads for 3-4 hours. Beads were then washed with Flag wash buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 300 mM NaCl, 10 mM KCl, 0.2% TritonX-100) 3 times, and then eluted 2 times in elution buffer (200ug/ml Flag peptide, 10 mM HEPES pH7.9, 0.1 M NaCl, 1.5 mM MgCl2, 0.05% TritonX-100. Trichloroacetic acid-precipitated protein mixtures from the Flag purifications were digested with endoproteinase Lys-C and trypsin (Roche) and analyzed by a 10-step MudPIT (Multidimensional Protein Identification Technology) on a LTQ linear ion trap mass spectrometer (Thermo Scientific) coupled to a Quaternary Agilent 1100 series HPLC pump and a nano-LC electrospray ionization source. Tandem mass (MS/MS) spectra were interpreted using ProluCID (10.1016/j.jprot.2015.07.001) against a database consisting of 44093 nonredundant human proteins (collated from Genome Reference Consortium Human Build 38 patch release 13 and NCBI RefSeq 2019-12-03 release), 426 usual contaminants (human keratins, IgGs, and proteolytic enzymes) and the shuffled sequences of each non-redundant entry to estimate false discovery rates. DTASelect (10.1021/pr015504q) and swallow, an in-house developed software (v. 0.0.1, https://github.com/tzw-wen/kite), were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here all controlled FDRs were less than 1%. DTASelect-filter.txt files (output from DTASelect software) were converted to mzIdentML with dtaselect2mzid (https://github.com/proteomicsyates/DTASelect2MzId). [doi:10.25345/C5Q75B] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: DYRK1A ; TRAF2 ; Sprouty 2 ; vesicle

Contact

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

Publications

Zhang P, Zhang Z, Fu Y, Zhang Y, Washburn MP, Florens L, Wu M, Huang C, Hou Z, Mohan M.
K63-linked ubiquitination of DYRK1A by TRAF2 alleviates Sprouty 2-mediated degradation of EGFR.
Cell Death Dis. 2021 Jun 11;12(6):608. doi: 10.1038/s41419-021-03887-2.

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