N-Glycolylneuraminic acid (Neu5Gc) is not normally detected in humans because humans lack the
hydroxylase enzyme that converts cytidine-5'-monophosphate-N-acetylneuraminic
acid (CMP-Neu5Ac) to
CMP-Neu5Gc; thus, any Neu5Gc appearing in the human body is aberrant. Neu5Gc has been observed in human
cancer cells and tissues. Moreover,
antibodies against Neu5Gc have been detected in healthy humans, which are obstacles to clinical
xenotransplantation and stem cell
therapies. Thus, the study of Neu5Gc in humans has important pathological and clinical relevance. Here, we report the N-glycoproteomics characterization of aberrant Neu5Gc in breast MCF-7
cancer cells and cancer stem cells (CSCs) at the molecular level of intact N-
glycopeptides, including comprehensive information (
peptide backbones, N-glycosites, N-
glycan monosaccharide compositions, and linkage structures) based on a target-decoy theoretical database search strategy and a spectrum-level false discovery rate (FDR) control ≤1%. The existence of Neu5Gc on N-
glycan moieties was further confirmed according to its characteristic
oxonium fragment
ions in the MS/MS spectra of either m/z 308.09816 (Neu5Gc) or 290.08759 (Neu5Gc-H2O). The results are an important addition to previously reported Neu5Ac data and can be further validated with targeted MS methods such as multiple and parallel reaction monitoring and biochemical methods such as immunoassays. This MS-based N-glycoproteomics method can be extended to the discovery and characterization of putative aberrant Neu5Gc in other
biological and clinical systems.