Simple Routes to Stable Isotope-Coded Native Glycans

• Published in: Analytical chemistry (Washington) Vol. 96; no. 1; pp. 163 - 169
• Main Authors: Helm, Johannes, Grünwald-Gruber, Clemens, Urteil, Jonathan, Pabst, Martin, Altmann, Friedrich
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 09.01.2024
• Subjects: Acetylation; Amino groups; Breast milk; Carbon; Galactose; Glycan; Hydrazine; Hydrazines; Isomers; Isotopes; N-glycans; Oligosaccharides; Polysaccharides; Reagents; Retention time; Sialic acids; Spectrometry; Stable isotopes
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.3c03446

Understanding the biological role of protein-linked glycans requires the reliable identification of glycans. Isomer separation and characterization often entail mass spectrometric detection preceded by high-performance chromatography on porous graphitic carbon. To this end, stable isotope-labeled glycans have emerged as powerful tools for retention time normalization. Hitherto, such standards were obtained by chemoenzymatic or purely enzymatic methods, which introduce, e.g., 13C-containing N-acetyl groups or galactose into native glycans. Glycan release with anhydrous hydrazine opens another route for heavy isotope introduction via concomitant de-N-acetylation. Here, we describe that de-N-acetylation can also be achieved with hydrazine hydrate, which is a more affordable and less hazardous reagent. Despite the slower reaction rate, complete conversion is achievable in 72 h at 100 °C for glycans with biantennary glycans with or without sialic acids. Shorter incubation times allow for the isolation of intermediate products with a defined degree of free amino groups, facilitating introduction of different numbers of heavy isotopes. Mass encoded glycans obtained by this versatile approach can serve a broad range of applications, e.g., as internal standards for isomer-specific studies of N-glycans, O-glycans, and human milk oligosaccharide by LC–MS on either porous graphitic carbon orfollowing permethylationon reversed phase.