Kiyonami R, Senko M, Zabrouskov V, Egertson J, Ting S , MacCoss M, Hühmer A.
ASMS 2013 Poster
Purpose: Develop a highly sensitive and highly selective data independent acquisition (DIA) workflow for large-scale targeted protein quantification on the new Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer.
Methods: For data independent acquisition set-up, three high-resolution, accurate-mass (HR/AM) selected ion monitoring (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 and throughput of this new approach were evaluated using various samples.
Results: The data collected from SIM scans with high resolving power provided unambiguous detection of targeted peptide peaks even at low concentration levels by separating interferences from matrix from peptide associated signal. Ten (10) attomole limits of detection (LODs) were observed for the isotopically labeled standards spiked in 500 ng E. coli digest matrix. Four (4) orders of linear dynamic range was observed with good precision. Highly reproducible and complete quantitative results were achieved by applying a targeted data extraction strategy after the data independent acquisition.
Thermo Fisher Scientific
United States
