MassIVE MSV000087028

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Quantitative Proteomic BioID Analysis of Zebrafish Hearts, Poss and Pronobis

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Description

Quantitative LC/MS/MS was performed on 3 uL of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) 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 in a data-dependent acquisition (DDA) mode of acquisition with an r=120,000 (@ m/z 200) full MS scan from m/z 375 - 1500 and a target AGC value of 2e5 ions with a 2 sec cycle time. Ion trap MS/MS scans were acquired with a Rapid scan rate, 100 ms max injection time, and a target AGC value of 5e3 ions. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours. Following 15 total UPLC-MS/MS analyses (excluding conditioning runs, but including 3 replicate QC injections; Table 1), data was imported into Proteome Discoverer 2.2 (Thermo Scientific Inc.), and analyses were aligned based on the accurate mass and retention time of detected ions (features) 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. The MS/MS data was searched against the TrEMBL D. rerio database (downloaded in May 2018) with additional proteins, including yeast ADH1, bovine serum albumin, as well as an equal number of reversed-sequence decoys) false discovery rate determination. Mascot Distiller and Mascot Server (v 2.5, Matrix Sciences) were utilized to produce fragment ion spectra and to perform the database searches. Database search parameters included fixed modification on Cys (carbamidomethyl) and variable modifications on Meth (oxidation) and Asn and Gln (deamidation). 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/C55F76] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: BioID, zebrafish

Contact

Principal Investigators:
(in alphabetical order) 		Ken Poss, Duke University, Dept of Cell Biology, USA
Submitting User: es3064
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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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