Computational protocol: Novel Insights into the PKCβ-dependent Regulation of the Oxidoreductase p66Shc*

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Protocol publication

[…] After the in vitro kinase assay, protein bands were excised from Coomassie-stained gel and digested with trypsin from porcine pancreas (Sigma-Aldrich) or Lys C (Sigma-Aldrich) as described previously (). Tryptic digests were analyzed using an UltiMate 3000 nano-HPLC system (Thermo Scientific, Bremen, Germany) coupled to a Q Exactive Plus or Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a Nanospray Flex ionization source. Settings different to Q Exactive Plus are bracketed. The peptides were separated on a homemade fused-silica microcapillary column (75 μm inner diameter × 280 μm outer diameter × 10 cm length) packed with 3-μm reverse-phase C18 material (Reprosil). Solvents for HPLC were 0.1% (v/v) formic acid (solvent A) and 0.1% (v/v) formic acid in 85% (v/v) 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 data-dependent mode selecting the top 20 most abundant isotope patterns with a charge >1 from the survey scan with an isolation window of 1.6 m/z. Survey full-scan MS spectra were acquired from 300–1750 m/z at a resolution of 60,000 with a maximum injection time of 120 ms and automatic gain control (AGC) target 1e6. The selected isotope patterns were fragmented by higher-energy collisional dissociation with normalized collision energy of 28 at a resolution of 15,000 with a maximum injection time of 120 ms and automatic gain control target 5e5.Data analysis was performed using Proteome Discoverer 1.4.1.14 (Thermo Scientific) with the search engine Sequest. The raw files were searched against a database (545 entries) containing the most common contaminants and p66. 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, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine were set as variable modifications. The peak area of the phosphorylated and non-phosphorylated peptides was calculated by summing up the peak areas or the corresponding precursor ions.Immunoprecipitated proteins were digested on beads with trypsin and analyzed using an UltiMate 3000 nano-HPLC system coupled via an electrospray ionization interface to an LTQ Velos mass spectrometer (Thermo Scientific). HPLC conditions were the same as with the in vitro kinase assay, except the particle size of the column material, which was 5-μm reverse-phase C18. The LTQ Velos mass spectrometer was operating in data-dependent mode selecting the top four most abundant isotope patterns with a charge >1 from the survey scan with an isolation window of 2.0 m/z. Survey full-scan MS spectra were acquired from 300–1800 m/z at an enhanced scan rate. Two-stage mass spectrometry (MS2) and MS3 (neutral loss from MS2) scans were fragmented by collision-induced dissociation with a normalized collision energy of 35. The settings for data analysis were the same as with the in vitro kinase assay, except for fragment mass tolerance, which was set to 0.8 Da. The amount of phosphorylated and non-phosphorylated peptide was calculated by the peak heights of the extracted ion chromatograms […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Chemicals Hydrogen Peroxide, Oxygen