We previously reported that N-glycans from bovine lung contain novel carboxylate groups. Here, we provide evidence that the carboxylated glycans contain glutamic acid. We labeled HeLa cells with [2,3-H-3]glutamate and used a carboxylate-specific monoclonal antibody to enrich for the desired proteins. PNGaseF digestion of these proteins released labeled N-glycans with a free amino group and 1-3 carboxylates. Mild acid hydrolysis had no effect, but strong acid hydrolysis of the glycans released > 80% of the 3 H as glutamate. Reducing the carboxylates to alcohols prior to hydrolysis eliminated the [H-3]glutamate and generated [H-3]4-amino 5-hydroxy pentanoic acid, suggesting that [3 H]glutamate was linked to the glycan through its gamma-carboxyl. The glutamate-containing N-glycans resisted exoglycosidase digestion and oligosaccharide processing inhibitors greatly reduced [3 H]glutamate incorporation. These results demonstrate that mammalian cells synthesize complex-type N-glycans with glutamate linked to their antennae, further expanding their potential for covalent or ionic interactions. (c) 2005 Elsevier Inc. All rights reserved.