MassIVE MSV000087774

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Calakos Shroff Nuclear Cytoplasm DYT-TOR1A proteomics

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Description

Quantitative LC/MS/MS was performed on 2 uL of each fraction, using a nanoAcquity UPLC system (Waters Corp.) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly, the fraction 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). Analytical separation was then 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 in 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 2e5 ions. MS/MS scans were acquired at Rapid scan rate (Ion Trap) with an AGC target of 5e3 ions and a max injection time of 100 milliseconds. The total cycle time for MS and MS/MS scans was 2 seconds. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each fraction injection was approximately 2 hours. Following 35 total UPLC-MS/MS analyses (excluding conditioning runs, but including 3 replicate QC Pool (TotalPool), 4 replicate nuclear (NucPool) and 4 replicate cytosolic Pool (CytoPool) injections), data was imported into Proteome Discoverer 2.2 (Thermo Scientific Inc.). Analyses were aligned based on the accurate mass and retention time of detected ions (features) using Minora Feature Detector algorithm in Proteome Discoverer. Protein levels are based on the relative peptide abundance measures which were calculated by area-under-the-curve (AUC) of the selected ion chromatograms of the aligned features across all runs. Levels are reported in arbitrary units (a.u.) Mascot Distiller and Mascot Server (v 2.5, Matrix Sciences) were utilized to produce fragment ion spectra from the MS/MS data and to perform database searches. The MS/MS data was searched against the SwissProt M. musculus database (downloaded in Apr 2017) to identify relevant M. musculus proteins. The MS/MS data was also searched against an equal number of reversed-sequence (decoys) for false discovery rate determination.. Database search parameters included fixed modification on Cys (carbamidomethyl) and , variable modifications on Meth (oxidation) and Asn and Gln (deamidation), full trypsin enzyme rules, and mass tolerances of 5ppm precursor ions and 0.8da product ions. Peptide Validator and Protein FDR Validator nodes in Proteome Discoverer were used to annotate the data at a maximum 1% protein false discovery rate. [doi:10.25345/C5W54T] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: proteomics, tor1a

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Principal Investigators:
(in alphabetical order) 		Nicole Calakos, Duke University, USA
Submitting User: es3064
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Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

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Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

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Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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