Stress granules proteome composition

ABSTRACT: Analysis of stress granules in HeLa cells, after exposure to different stimuli. Granules were collected by a newly devised enrichment method, described in manuscript. Proteomics sample preparation of granules was carried out using the Single-pot, solid phase-enhanced sample preparation, SP3 method (Hughes CS et al. Nat Protoc 14, 68 85 (2019)). Desalting, clean-up and loading to the MS was done using an Evosep one EV1000 instrument, following manufacturers protocol, but loading of sample to Evotips was done a pre-existing volume of A Buffer. Samples were measured on a Thermo Exploris 480 mass spectrometer, using data-independent acquisition (DIA) (30 SPD, 44 LC min gradient (Evosep), MS1: 120000 res, 350-1400 m/z, inj. time 50 ms, MS2: 14 m/z isolation windows, 1 /mz overlap, precursor mass range. 350-1020 m/z, stepped collision energy 25,27.5,30, 22 ms inj. time. RAW files were analyzed using Spectronaut v16, using directDIA mode with default settings, using as database the Homo Sapiens EBI Reference proteome, downloaded on 03/2021 and including an in-house curated contaminants list based on the MaxQuant's contaminants list. Protein summarisation was done with MSstats, using default settings.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Gerhard Mittler  

PROVIDER: MSV000090634 | MassIVE | Wed Nov 02 17:47:00 GMT 2022

SECONDARY ACCESSION(S): PXD037887

REPOSITORIES: MassIVE