For gel band analysis, SDS-PAGE gel bands were subjected to reduction, alkylation, and in-gel tryptic digestion as described here: https://genome.duke.edu/sites/genome.duke.edu/files/In-gelDigestionProtocolrevised_0.pdf. Digested peptides were lyophilized to dryness and resuspended in 12 uL of 0.2% formic acid/2% acetonitrile. Each sample was subjected to chromatographic separation on a Waters NanoAquity UPLC equipped with a 1.7 um HSS T3 C18 75 um I.D. x 250 mm reversed-phase column (NanoFlow data). The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. 3 uL was injected and peptides were trapped for 3 min on a 5 um Symmetry C18 180 um I.D. x 20 mm column at 5 ul/min in 99.9% A. The analytical column was then switched in-line and a linear elution gradient of 5% B to 40% B was performed over 30 min at 400 nL/min. The analytical column was connected to a Fusion Lumos mass spectrometer (Thermo) through an electrospray interface operating in a data-dependent mode of acquisition. The instrument was set to acquire a precursor MS scan from m/z 375-1500 at R=120,000 (target AGC 2e5, max IT 50 ms) with MS/MS spectra acquired in the ion trap (target AGC 5e3, max IT 100 ms). For all experiments, HCD energy settings were 30v and a 20 s dynamic exclusion was employed for previously fragmented precursor ions.
Raw LC-MS/MS data files were processed in Proteome Discoverer (Thermo Scientific) and then submitted to independent Mascot searches (Matrix Science) against a Human protein database containing both forward (20260 entries) and reverse entries of each protein. Search tolerances were 5 ppm for precursor ions and 0.8 Da for product ions using trypsin specificity with up to two missed cleavages. Carbamidomethylation (+57.0214 Da on C) was set as a fixed modification, whereas oxidation (+15.9949 Da on M) and deamidation (+0.98 Da on NQ) were considered a dynamic mass modifications. All searched spectra were imported into Scaffold (v4.4, Proteome Software) and scoring thresholds were set to achieve a peptide false discovery rate of 1% using the PeptideProphet algorithm.
[doi:10.25345/C58V5H]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Her2, Abl, LCMS
Principal Investigators: (in alphabetical order) |
Ann Marie Pendergast, Duke University, USA |
Submitting User: | es3064 |
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