Computational protocol: Identification of potential novel interaction partners of the sodium-activated potassium channels Slick and Slack in mouse brain

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Protocol publication

[…] For mass spectrometric sequencing, eluates obtained from immunoprecipitation experiments were run on SDS-PAGE minigels and stained with Coomassie blue R250 (Biorad). Protein bands were excised from gel and digested with trypsin from porcine pancreas (Sigma-Aldrich, Vienna, Austria) as previously described . Tryptic digests were analyzed using an UltiMate 3000 nano-HPLC system (Thermo Scientific, Germering, Germany) coupled to a Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with a Nanospray Flex ionization source. The peptides were separated on a homemade fritless fused-silica microcapillary column (75 µm i.d.×280 µm o.d.×10 cm length) packed with 3 µm reversed-phase C18 material (Reprosil). Solvent for HPLC were 0.1% formic acid (solvent A) and 0.1% formic acid in 85% acetonitrile (solvent B). The gradient profile was as follows: 0–2 min, 4% B; 2–55 min, 4–50% B; 55–60 min, 50–100% B, and 60–65 min, 100% B. The flow rate was 250 nL/min.The Q Exacitve Plus mass spectrometer was operating in the data dependent mode selecting the top 12 most abundant isotope patterns with charge >1 from the survey scan with an isolation window of 1.6 mass-to-charge ratio (m/z). Survey full scan MS spectra were acquired from 300 to 1750m/z at a resolution of 70,000 with a maximum injection time (IT) of 120 ms, and automatic gain control (AGC) target 1e6. The selected isotope patterns were fragmented by higher-energy collisional dissociation (HCD) with normalized collision energy of 25 at a resolution of 17,000 with a maximum IT of 120 ms, and AGC target 5e5.Data Analysis was performed using Proteome Discoverer 1.4.1.14 (Thermo Scientific) with search engine Sequest. The raw files were searched against the mus musculus database (167,940 entries) extracted from the NCBInr database released on June 2, 2014. Precursor and fragment mass tolerance was set to 10 ppm and 0.02 Da, respectively, and up to two missed cleavages were allowed. Carbamidomethylation of cysteine, and oxidation of methionine were set as variable modifications. Peptide identifications were filtered at 1% false discovery rate. [...] Protein sequences of different species were obtained from Pubmed. Alignments and creation of percent identity matrix were performed using Clustal Omega (rel. 1.2.1) with default settings. Topology of mouse TMEM 263 was analyzed using the web based protein structure prediction program TMHMM server (rel. 2.0).Pubmed accession numbers of protein sequences used:Rattus norvegicus: gi|157823853; Mus musculus: gi|81881684; Homo sapiens gi|74730713; Xenopus laevis gi|82186192; Danio rerio gi|82188197; Bos taurus gi|77736291; Gallus gallus gi|57525414; Pan pansicus gi|675800328 […]

Pipeline specifications

Software tools Clustal Omega, TMHMM
Application Membrane protein analysis
Organisms Mus musculus, Homo sapiens
Chemicals Potassium, Sodium