Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.

Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification.