Dataset Information

Nicchitta SILAC RNA binding Proteins

ABSTRACT: Quantitative LC-MS/MS was performed on 2.5 uL (6%) 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) equipped with a FAIMSPro 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/acetonitrile), 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 (40v, 60v, 80v). 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 in the Orbitrap at r=50,000 (m/z 200) from m/z 100 with a target AGC value of 1e5 and max fill time of 35 ms. The total cycle time for each CV was 1s, with total cycle times of 3 sec between like full MS scans. A 45s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each fraction injection was approximately 2 hours. Quantitative LC-MS/MS Analysis. Next, data was imported into Proteome Discoverer 2.5 (Thermo Scientific Inc.) and all LC-MS/MS runs were aligned based on the accurate mass and retention time of detected ions (features) which contained MS/MS spectra using Minora Feature Detector algorithm in Proteome Discoverer. Relative peptide abundance was calculated based on area under the curve (AUC) of the selected ion chromatograms of the aligned features across all runs.

INSTRUMENT(S): Orbitrap Fusion Lumos

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

SUBMITTER: Dr. Christopher Nicchitta