Analysis of Sialic Acid Linkage in N-Linked Glycopeptides Using Liquid Chromatography–Electron-Activated Dissociation Time-of-Flight Mass Spectrometry

Researchers from Sciex, Concord, Ontario, Canada and Redwood City, California, have developed a groundbreaking method for analyzing sialic acid (Neu5Ac, Sa) linkage in N-linked glycans within glycopeptides. The study, published in the journal Analytical Chemistry, introduces a novel liquid chromatography-tandem mass spectrometry approach that eliminates the need for sialic acid derivatization (1).

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Led by Takashi Baba, the research team established a reversed-phase high-performance liquid chromatography (HPLC) separation method utilizing mobile phases with a higher concentration of formic acid. This approach successfully differentiated N-glycopeptides based on the Sa linkage. Additionally, the scientists introduced an innovative characterization technique for Sa linkages in N-glycopeptides using electron-activated dissociation.

By employing hot electron capture dissociation with an electron beam energy above 5 eV, the researchers cleaved the glycosidic bonds in glycopeptides. This process resulted in the breaking of glycosidic bonds on both sides of the oxygen atom within the glycan antennas. The cleavage at the reducing end, known as C-type ion fragmentation, allowed for differentiation between Sa-Gal, Gal-GlcNAc, and GlcNAc-Man linkages. The team proposed a rule for characterizing Sa linkages based on the Sa-Gal products.

To demonstrate the applicability of their method, the researchers applied it to N-glycopeptides obtained from a tryptic fetuin digest. The optimized reversed-phase HPLC separation facilitated the successful identification of numerous isomeric glycoforms within the glycopeptides, each exhibiting different Sa linkages. Simultaneously, the peptide backbones were sequenced using hot electron capture dissociation.

The novel liquid chromatography-electron-activated dissociation time-of-flight mass spectrometry approach presented in this study offers a significant advancement in the analysis of Sa linkages in N-linked glycans within glycopeptides.

The findings of this study open up new possibilities for in-depth investigations into the role of sialic acid linkages in glycoproteins and their implications in various biological processes. The researchers envision that this innovative method will contribute to a deeper understanding of glycosylation patterns and their significance in health and disease. By eliminating the need for sialic acid derivatization, the method streamlines the analytical process, providing researchers with a more efficient and accurate tool for glycoproteomics research.

Reference

(1) Liu, S.; Ryumin, P.; Albanese, J.; Zhang, Z.; Baba, T. Analysis of Sialic Acid Linkage in N-Linked Glycopeptides Using Liquid Chromatography–Electron-Activated Dissociation Time-of-Flight Mass Spectrometry. Anal. Chem. 2023, 95 (19), 7458-7467. DOI: https://doi.org/10.1021/acs.analchem.2c04581