Dataset Information

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

ABSTRACT: 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).

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Laurence Florens

PROVIDER: MSV000085815 | MassIVE | Thu Jul 23 10:14:00 BST 2020

SECONDARY ACCESSION(S): PXD020533

REPOSITORIES: MassIVE

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Publications

K63-linked ubiquitination of DYRK1A by TRAF2 alleviates Sprouty 2-mediated degradation of EGFR.

Zhang Pengshan P Zhang Zhe Z Fu Yinkun Y Zhang Ying Y Washburn Michael P MP Florens Laurence L Wu Min M Huang Chen C Hou Zhaoyuan Z Mohan Man M

Cell death & disease 20210611 6

Dual specificity tyrosine phosphorylation regulated kinase 1A, DYRK1A, functions in multiple cellular pathways, including signaling, endocytosis, synaptic transmission, and transcription. Alterations in dosage of DYRK1A leads to defects in neurogenesis, cell growth, and differentiation, and may increase the risk of certain cancers. DYRK1A localizes to a number of subcellular structures including vesicles where it is known to phosphorylate a number of proteins and regulate vesicle biology. Howeve ...[more]

PMID: 34117217

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