Computational protocol: Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster

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Protocol publication

[…] For immunostaining 0.8×106 cells were seeded onto 16 mm coverslips and allowed to attach for 1 h at RT. Cells were washed briefly with PBS and fixed in 2% (v/v) formaldehyde in PBS for 7.5 min on ice. Cells were permeabilized in PBS, 0.25% (v/v) Triton X-100, 1% (v/v) formaldehyde for additional 7.5 min on ice. Afterwards, cells were washed with PBS and blocked in PBS containing 3% (w/v) BSA for 1 h at RT. Fixed cells were incubated with suitable primary antibody diluted in blocking solution (PBS, 2% BSA, 0.01% Triton X-100, 1.2% Normal Donkey Serum) for 1 h at RT. After two wash steps cells were incubated for an additional hour with suitable secondary antibody at RT. Cells were washed again before DNA counterstaining with DAPI (1 μg/μl in PBS) for 2 min. Cells were washed twice with PBS and mounted using 10 μl Vectashield mounting medium. The coverslip was sealed to the object slide by nail polish. Immunofluorescence was detected using a Zeiss Axiovert 200 epifluorescence microscope equipped with a CDD Camera (AxioCamMR, Zeiss). Images were level-adjusted in FIJI and edited using Adobe Photoshop CS5 and Adobe Illustrator CS5. [...] Peptide fractions were analyzed on a quadrupole Orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific) equipped with a UHPLC system (EASY-nLC 1000, Thermo Scientific) as described []. Peptide samples were loaded onto C18 reversed phase columns (15 cm length, 75 μm inner diameter, 1.9 μm bead size) and eluted with a linear gradient from 8 to 40% acetonitrile containing 0.1% formic acid in 2 hours. The mass spectrometer was operated in data dependent mode, automatically switching between MS and MS2 acquisition. Survey full scan MS spectra (m/z 300–1700) were acquired in the Orbitrap. The 10 most intense ions were sequentially isolated and fragmented by higher-energy C-trap dissociation (HCD). An ion selection threshold of 5,000 was used. Peptides with unassigned charge states, as well as with charge states less than +2 were excluded from fragmentation. Fragment spectra were acquired in the Orbitrap mass analyzer. Raw data files were analyzed using MaxQuant (development version 1.5.2.8) []. Parent ion and MS2 spectra were searched against a database containing Drosophila melanogaster protein sequences obtained from the UniProtKB released in May 2016 using Andromeda search engine []. Spectra were searched with a mass tolerance of 6 ppm in MS mode, 20 ppm in HCD MS2 mode, strict trypsin specificity and allowing up to 3 miscleavages. Cysteine carbamidomethylation was searched as a fixed modification, whereas protein N-terminal acetylation, methionine oxidation, n-ethylmaleimide modification of cysteines (mass difference to cysteine carbamidomethylation) and di-glycine-lysine were searched as variable modifications. Site localization probabilities were determined by MaxQuant using the PTM scoring algorithm as described previously []. The dataset was filtered based on posterior error probability (PEP) to arrive at a false discovery rate of below 1% estimated using a target-decoy approach []. Di-glycine lysine modified peptides with a minimum score of 40 and delta score of 6 are reported and used for the analyses. […]

Pipeline specifications

Software tools Adobe Illustrator, MaxQuant, Andromeda
Databases UniProtKB
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Drosophila melanogaster, Homo sapiens