BioID-MS of Helios point mutations and wildtype, all MAC-tagged C-terminally.
Included mutations:
I325V
M301K
R106W
R291X
L328F
WT
V347M
Y359C
Y200C
P18S
N220S
Analysis was performed on a Q-Exactive mass spectrometer with an EASY-nLC 1000 Liquid Chromatograph Q Exactive Hybrid Quadrupole-Orbitrap system via an electrospray ionization sprayer (Thermo Fisher Scientific), using Xcalibur version 3.0.63. Peptides were eluted from the sample with a C18 precolumn (Acclaim PepMap 100, 75 um x 2 cm, 3 um, 100 A; Thermo Scientific) and analytical column (Acclaim PepMap RSLC, 65 um x 15 cm, 2 um, 100 A; Thermo Scientific), using a 60 minute buffer gradient ranging from 5 to 35% Buffer B, then a 5 min gradient from 35 to 80% Buffer B and 10 minute gradient from 80 to 100% Buffer B (0.1% formic acid in 98% acetonitrile and 2% HPLC grade water). 4 ul of peptide sample was loaded by by an cooled autosampler. Data-dependent FTMS acquisition was in positive ion mode for 80 min. A full scan (200-2000 m/z) was performed with a resolution of 70000 followed by top10 CID-MS2 ion trap scans with a resolution of 17500. Dynamic exclusion was set for 30 s. Database search was performed with Proteome Discoverer 1.4 (Thermo Scientific) using the SEQUEST search engine on the Reviewed human proteome in UniProtKB/SwissProt databases (http://www.uniprot.org, downloaded Nov. 2019). Trypsin was selected as the cleavage enzyme and maximum of 2 missed cleavages were permitted, precursor mass tolerance at +-15 ppm and fragment mass tolerance at 0.05 Da. Carbamidomethylation of cysteine was defined as a static modification. Oxidation of methionine and for BioID samples biotinylation of lysine and N-termini were set as variable modifications.
[doi:10.25345/C59C37]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Helios ; BioID
Principal Investigators: (in alphabetical order) |
Markku Varjosalo, University of Helsinki, Finland |
Submitting User: | Smoosh |
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