MassIVE MSV000092789

Partial Public PXD044965

ALK signalling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition

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Description

Phosphoproteomics DIA LC-MSMS analysis Phospho-enriched peptides were resuspended in aqueous formic acid and 0.2 ug of peptides subjected to LC-MS/MS analysis using an Orbitrap Exploris 480 Mass Spectrometer fitted with an Vanquish Neo (both Thermo Fisher Scientific) and a custom-made column heater set to 60C. Peptides were resolved using a RP-HPLC column (75um x 30cm) packed in-house with C18 resin (ReproSil-Pur C18-AQ, 1.9 um resin; Dr. Maisch GmbH) at a flow rate of 0.2 uLmin-1. Separation of peptides was achieved using the following gradient: 4% Buffer B to 10% Buffer B in 5 min, 10% Buffer B to 35% Buffer B in 45 min, 35% Buffer B to 50% Buffer in 10 min. Buffer A was 0.1% formic acid in water and buffer B was 80% acetonitrile, 0.1% formic acid in water. The mass spectrometer was operated in DIA acquisition mode with a total cycle time not exceeding approximately 3 s. For MS1, the following parameters were set: Resolution: 120,000 FWHM (at 200 m/z), Scan Range: 350-1400 m/z, Injection time: 25 ms, Normalized AGC Target: 300%. MS2 (SWATH) scans were acquired using the following parameters: Isolation Window: 8 m/z, HCD Collision Energy (normalized): 28%, Normalized AGC target: 1000%, Resolution: 15,000 FWHM (at 200 m/z), Precursor Mass Range: 400 - 1200 m/z, Max. Fill Time: 22 ms, DataType: Centroid. In total 100 DIA (MS2) mass windows per MS cycle followed by the one MS1 scan. Data analysis of phosphoproteomics DIA-MS data The acquired raw files were searched using SpectroNaut (v17.1, PTM workflow, default settings) against a human database (consisting of 20360 protein sequences downloaded from Uniprot on 20220222) and 392 commonly observed contaminants using the following search criteria: full tryptic specificity was required (cleavage after lysine or arginine residues, unless followed by proline); 3 missed cleavages were allowed; carbamidomethylation (C) was set as fixed modification; oxidation (M), N-acetlyation (N-term) and phosphorylation (STY) were applied as variable modifications. The normalized, phosphosite centric and statstically analysed quantiative results were exported as a csv-table from the SpectroNaut software using the candidate list export option without any filters applied. [doi:10.25345/C5H98ZQ28] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: alk ; signalling

Contact

Principal Investigators:
(in alphabetical order) 		Alexander Schmidt, Biozentrum, Universtiy of Basel, 4056 Basel, Switzerland, N/A
Ruth Palmer, University of Gothenburg, Sweden
Submitting User: trendsetter
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