Mass spectrometric peptide analysis of 2 DE ‐separated mouse spinal cord and rat hippocampus proteins suggests an NG x G motif of importance for in vivo deamidation

Asparagine deamidation is a common nonenzymatic post‐translational modification comprising the conversion of asparaginyl residues to aspartyl and isoaspartyl residues, respectively. As a result an additional negative charge is introduced that can affect the tertiary structure as well as the biological activity of a protein. Since deamidation reduces the protein's p I value, differentially deamidated forms of a protein can be separated in 2 D gels. We have analyzed a dataset of 430 protein spots from 2 D gels that contained mouse spinal cord proteins and estimated that roughly 10% of the spots in a C oomassie‐stained gel derive from in vivo deamidation at particular asparaginyl residues. Several of the deamidated protein forms, e.g. tropomodulin‐2, V ‐type proton ATP ase subunit B , and protein disulfide‐isomerase A 3 were also found in 2 D gels of proteins extracted from rat hippocampus. All identified deamidation sites contained a glycine residue on the carboxyl side of the asparaginyl residue. Strikingly, a second glycine residue at the +3 position was found in the majority of the deamidated peptides. We propose that the NG x G motif confers exceptional susceptibility to in vivo asparagine deamidation.