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 pI value, differentially deamidated forms of a protein can be separated in 2D gels. We have analyzed a dataset of 430 protein spots from 2D gels that contained mouse spinal cord proteins and estimated that roughly 10% of the spots in a Coomassie-stained gel derive from in vivo deamidation at particular asparaginyl residues. Several of the deamidated protein forms, e.g. tropomodulin-2, V-type proton ATPase subunit B, and protein disulfide-isomerase A3 were also found in 2D 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 NGxG motif confers exceptional susceptibility to in vivo asparagine deamidation.