Mapping sites of O-fucose modification on mouse NOTCH2

The O-fucose modification sites on mouse NOTCH2 were determined by mass spectral glycoproteomic site mapping. The Extracellular Domain of mouse NOTCH2 was produced in HEK293T cells in the presence or absence of the three Fringe enzymes: Lunatic fringe, Manic fringe, or Radical fringe. The protein was purified from the medium, reduced and alkylated, digested with trypsin, chymotrypsin or V8 protease, and the resulting peptides were analyzed by nano-LC mass spectrometry. The majority of the sites were identified using an Agilent model 6340 Ion Trap mass spectrometer. A few sites were identified using a Q-Exactive Plus mass spectrometer.