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 (13)C 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.