MassIVE MSV000086045

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Thiele HSF1 C HS HSR Interactome

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Description

Methods. Quantitative LCMSMS was performed on 4 uL of each sample using a nanoAcquity UPLC system Waters Corp coupled to a Thermo QExactive HF 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 vv 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 90min linear gradient of 5 to 30 acetonitrile with 0.1 percent formic acid at a flow rate of 400 nanoliters minute nL min with a column temperature of 55C. Data collection on the QExactive HF mass spectrometer was performed in a data dependent acquisition DDA mode of acquisition with a r 120,000 mz 200 full MS scan from mz 375 to 1600 with a target AGC value of 3e6 ions followed by 15 MSMS scans at r 30000 mz 200 at a target AGC value of 5e4 ions and 45 ms. 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 19 total LCMSMS analyses excluding conditioning runs, but including 4 replicate QC injections data was imported into Rosetta Elucidator v 4.0 Rosetta Biosoftware Inc., and analyses were aligned based on the accurate mass and retention time of detected ions features using PeakTeller algorithm in Elucidator. 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 MSMS data was searched against the SwissProt M. musculus database downloaded in May 2017 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. Full tryspin enzyme rules were used with up to 2 missed cleavages with mass tolerances of 5 ppm for precursor and 0.02da for product ions. After individual peptide scoring using the PeptideProphet algorithm in Elucidator, the data was annotated at a 1 percent peptide false discovery rate. [doi:10.25345/C5KF4S] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: HSF interactome, HF

Contact

Principal Investigators:
(in alphabetical order) 		Erik Soderblom, Duke Univ, 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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