Computational protocol: GSK3β-Dzip1-Rab8 Cascade Regulates Ciliogenesis after Mitosis

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Protocol publication

[…] All analyses were performed on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) at a resolution of 60,000. For nano-liquid chromatography, Eksigent nanoLC 1D plus systems were equipped with 2-mm ReproSil-Pur C18-AQ (Dr. Maisch) trapping columns (packed in house; i.d., 1,150 μm; resin, 5 μm) and 200-mm ReproSil-Pur C18-AQ (Dr. Maisch) analytical columns (packed in house; i.d., 75 μm; resin, 3 μm). The solvents used were 0.5% formic acid-water solution (buffer A) and 0.5% formic acid-acetonitrile solution (buffer B). Trapping was performed at 2 μl/min buffer A for 15 min, and elution was achieved with a gradient of 0%–32% buffer B over 80 min, 32%–50% buffer B over 6 min, and 80% buffer B over 6 min at a flow rate of 300 nl/min. Eluting peptide cations were converted to gas-phase ions by a Nanospray Flex (Thermo Scientific) ion source at 2.0 kV. The mass spectrometer was operated in the data-dependent mode to automatically switch between mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Survey full-scan MS spectra were acquired from m/z 300 to m/z 1,800, and the ten most intense ions with a charge state above 2 and above an intensity threshold of 500 were fragmented in the linear ion trap using a normalized collision energy of 35%. For the orbitrap, the AGC target value was set at 1 × 106, and the maximum fill time for full MS was set at 500 ms. Fragment ion spectra were acquired in the LTQ Orbitrap XL with an AGC target value of 3 × 104 and a maximum fill time of 150 ms. Dynamic exclusion for selected precursor ions was set at 90 s. The lock mass option was enabled for the 462.14658 ion. The raw data were processed using Proteome Discoverer (version 1.4.0.288, Thermo Fisher Scientific). MS2 spectra were searched with the Sequest HT engine against the UniProt mouse complete proteome database (release 2013_06, 50,790 protein sequences). The database was searched with the following parameters: precursor mass tolerance, 20 ppm; MS/MS mass tolerance, 0.6 Da; two missed cleavages for tryptic peptides; dynamic modification oxidation (M), phosphorylation (STY); static modification carbamidomethylation (C). Peptide spectral matches were validated by a targeted decoy database search at 1% false discovery rate. With Proteome Discoverer, peptide identifications were grouped into proteins according to the law of parsimony. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Chemicals Sucrose