Project PXD035503: "The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis"

Data collection method:

HEK293T cells were synchronised in late G2 and mitosis as described above. Four μg of IgG or of a HECTD1 antibody (Ab101992) were coated onto Dynabead magnetic beads and incubated with either lysates from G2 or M-phase synchronised cells for 1 hr at RT prior to washes, and denaturation in 2xLDS sample buffer/100 mM DTT at 95°C for 5 min. Samples were then loaded onto a 4-12% Bis-Tris SDS polyacrylamide gel which was then stained with Imperial Protein Stain (ThermoFisher Scientific). Gel lanes were sliced into of 1-2 mm2 pieces and processed by in-gel trypsin digestion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an LTQ Orbitrap Velos (ThermoFisher Scientific) mass spectrometer coupled to an Ultimate 3000 nano-ultra performance liquid chromatography (UPLC) system (Dionex, Sunnyvale, CA, USA). The mass spectrometer was operated in data-dependent acquisition mode using a Top10 method and a 32-minute liquid chromatography gradient. The MS1 survey scans were acquired in the Orbitrap at a resolution of 30,000 and an m/z range of 300-2,000. MS2 data were acquired in the Ion Trap and precursor ion isolation was performed with an isolation width of 2.0 Da and CID activation with a normalised collision energy of 35.

Data processing and preparation activities:

The LC-MS/MS data were searched with the MASCOT search algorithm [1]. Mascot distiller was used to produce Mascot compatible (.mgf) files from the raw LC-MS/MS data. Mascot (Matrix Science, algorithm version 2.2.04) was used to perform database searches of the Uniprot database without taxonomy restriction (14,423,061 entries). Variables were set to: 2+ and 3+ ions, peptide mass tolerance 10 ppm, fragment mass tolerance 0.8 Da, number of missed cleavages: one, instrument type: ESI-TRAP, variable modifications: Carbamidomethylation (Cys), Oxidation (Met). N-terminal pyroglutamate (Gln->pyroGly), phosphorylation (Ser, Thr, Tyr). MS/MS data were further validated using Scaffold (http://www.proteomesoftware.com). [1] Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data., Electrophoresis. 20, 3551-3567.