AUTHOR=Yang Wenqian , Jiang Yuan , Guo Qulian , Tian Zhixin , Cheng Zhigang TITLE=Aberrant N-glycolylneuraminic acid in breast MCF-7 cancer cells and cancer stem cells JOURNAL=Frontiers in Molecular Biosciences VOLUME=9 YEAR=2022 URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2022.1047672 DOI=10.3389/fmolb.2022.1047672 ISSN=2296-889X ABSTRACT=

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.