The identification of bacterial metabolites produced by the microbiota is a key point to understand its role in human health. Among them, lipo-amino acids (LpAA), which are able to cross the epithelial barrier and to act on the host, are poorly identified. Structural elucidation of few of them was performed by high-resolution tandem mass spectrometry based on electrospray combined with selective ion dissociations reach by collision-induced dissociation (CID). The negative ions were used for their advantages of yielding only few fragment ions sufficient to specify each part of LpAA with sensitivity. To find specific processes that help structural assignment, the negative ion dissociations have been scrutinized for an LpAA: the N-palmitoyl acyl group linked to glutamic acid (C16Glu). The singular behavior of [C16Glu-H]¯ towards CID showed tenth product ions, eight were described by expected fragment ions. In contrast, instead of the expected product ions due to CONH-CH bond cleavage, an abundant complementary dehydrated glutamic acid and fatty acid anion pair were observed. Specific to glutamic moiety, they were formed by a stepwise dissociation via molecular isomerization through ion-dipole formation prior to dissociation. This complex dissociated by partner splitting either directly or after inter-partner proton transfer. By this pathway, surprising regeneration of deprotonated fatty acid takes place. Such regeneration is comparable to that occurred from dissociation to peptides containing acid amino-acid. Modeling allow to confirm the proposed mechanisms explaining the unexpected behavior of this glutamate conjugate.