Quantitative LC-MRM was performed on 1 ug of protein digest spiked with 10 fmol of 173 SIL peptides using a nanoAcquity UPLC system (Waters Corp) coupled to a Waters Xevo TQ-XS triple quadrupole mass spectrometer via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 300 mm x 180 mm 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 150 mm column (Waters Corp.) using a 55-min gradient of 5 to 40% 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 Xevo TQ-XS mass spectrometer was performed in a targeted mode following method creation within Skyline (MacCoss Laboratory, Univ of Washington) with retention time scheduling set to 4 min around the average peak apex retention time from three SIL peptide alone acquisitions. Average peak widths were set to 20 seconds with 12 points across the peak, the auto-dwell feature was enabled, and the optimal CE for each precursor was calculated experimentally from a SIL peptide alone analysis.
[doi:10.25345/C53F4KX9X]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Tageted SRM
Principal Investigators: (in alphabetical order) |
Virginia Kraus, Duke University, USA |
Submitting User: | es3064 |
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