Ryan D. Bomgarden1; Katherine A. Overmyer2; Elyse C. Freiberger2; Alexander S. Hebert2; Michael S. Westphall2; Juergen H. Cox3; Joshua J. Coon2; John C. Rogers1
International Hupo 2016
Purpose: To expand multiplex quantitation of stable isotope labeling using amino acids in cell culture (SILAC) using neutron encoded amino acids
(NeuCode™) from 2-plex up to 7-plex.
Methods: A549 and HepG2 cells (ATCC) were cultured with SILAC media containing 10% dialyzed FBS and one of 8 different lysine isotopologs
K000, K202, K040, K602, K341, K080, K642, and K390 (KXXX is lysine with designated number of 13C, 2H, and 15N, respectively). Samples were
combined for multiplex analysis using a Thermo Scientific™ Orbitrap™ Fusion or Orbitrap Elite mass spectrometer operating at an MS1 resolving
power of 500K at m/z 200 and 480K at m/z 400, respectively. MaxQuant software modified for NeuCode data analysis was used for peptide
identification and quantification.
Results: Cells cultured with the different lysine isotopologs exhibited similar viability and rate of heavy amino acid incorporation for two human cell
lines. High resolution MS1 scans using Orbitrap technology were used to separate near isobaric lysine isotopolog isotopic clusters at +4, +8, and +12
Da. A modified version of MaxQuant software was used to automatically extract the quantitative signatures.
1 Thermo Fisher Scientific, Rockford, IL, USA;
2 University of Wisconsin Madison, Madison, WI USA;
3 Max Planck Institute of Biochemistry, Martinsried, Germany
United States
