ATG9A interactome using BioID proximity labeling
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ABSTRACT: Quantitative LC/MS/MS was performed on 2 uL of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipied with a FAIMS device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/ac etonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40, 60, 80). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (@ m/z 200) full MS scan from m/z 375-1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired at Rapid scan rate (Ion Trap) with an AGC target of 1e4 ions and a max injection time of 100 ms. The total cycle time for each CV was 0.66s, with total cycle times of 2 sec between like MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours.
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: Josh Anderson
PROVIDER: MSV000087520 | MassIVE | Wed May 26 07:08:00 BST 2021
REPOSITORIES: MassIVE
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