Accurate Identification of Deamidated Peptides in Global Proteomics Using a Quadrupole Orbitrap Mass Spectrometer

Deamidation of asparagine and glutamine residues is a common post-translational modification. Researchers often rely on mass spectrometric based proteomic techniques for the identification of these post-translational sites. Mass spectral analysis of deamidated peptides is complicated and often misassigned due to overlapping 13C peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by 19.34 mDa. For proper assignment, it is inherently important to use a mass spectrometer with high mass measurement accuracy and high resolving power. Herein, mouse brain tissue lysate was prepared using filter-aided sample preparation (FASP) method and Stage Tip fractionation followed by analysis on a nanoLC coupled with a quadrupole orbitrap (Q-Exactive) mass spectrometer to accurately identify more than 5400 proteins. Mass spectral data was processed using MASCOT and ProteoIQ for accurate identification of peptides and proteins. MASCOT search values for precursor and MS/MS mass tolerances were investigated, and it was determined that data searched with greater than 5 ppm precursor mass tolerance resulted in the misassignment of deamidated peptides. Peptides that were identified with a mass measurement accuracy of ±5 ppm were correctly assigned.