Kiyonami R, Patel B, Senko M, Zabrouskov V, Egertson J, Ting S, MacCoss M, ROgers J, Huhmer A.
ASMS 2014 Poster
Purpose: Evaluate the quantitative performances for large scale targeted protein experiments using the unique data-independent acquisition workflow (WiSIM) which uses high resolution accurate mass of SIM scans for quantification and CID MS/MS spectra of MS/MS scans for simultaneous peptide sequence confirmation on a Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer.
Methods: Orbitrap Fusion MS equipped with a Thermo Scientific™ EASY-Spray™ source is used for all experiments. For data-independent acquisition set up, three HRAM SIM scans (240,000 FWHM) with wide isolation windows (200 amu) were used to cover all precursor ions of 400–1000 m/z. In parallel with each SIM scan, 17 sequential ion trap MS/MS with 12 amu isolation windows were acquired to cover the associated 200 amu SIM mass range. Quantitative information for all precursor ions detected in three sequential SIM scans is recorded in a single run. Plus, all MS/MS fragment information over the mass range of 400–1000 m/z is recorded for sequence confirmation of any peptide of interest by querying specific fragment ions in the spectral library. The quantitative performances using this approach for large scale targeted protein quantification were evaluated using various samples.
Results: The data collected from SIM scans with extreme high resolving power provided high mass accuracy, which allowed unambiguous detection of targeted peptide peaks for highly reproducible and accurate quantitative results over thousands of peptides by applying targeted data extraction strategy after the data independent acquisition.
Thermo Fisher Scientific, University of Washington.
United States
