1. Capillary electrophoresis-mass spectrometry for direct structural identification of serum N-glycans.
- Author
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Snyder CM, Zhou X, Karty JA, Fonslow BR, Novotny MV, and Jacobson SC
- Subjects
- Humans, Isomerism, N-Acetylneuraminic Acid, Polysaccharides chemistry, Pyrenes, Blood Chemical Analysis methods, Electrophoresis, Capillary, Mass Spectrometry, Polysaccharides blood, Polysaccharides isolation & purification
- Abstract
Through direct coupling of capillary electrophoresis (CE) to mass spectrometry (MS) with a sheathless interface, we have identified 77 potential N-glycan structures derived from human serum. We confirmed the presence of N-glycans previously identified by indirect methods, e.g., electrophoretic mobility standards, obtained 31 new N-glycan structures not identified in our prior work, differentiated co-migrating structures, and determined specific linkages on isomers featuring sialic acids. Serum N-glycans were cleaved from proteins, neutralized via methylamidation, and labeled with the fluorescent tag 8-aminopyrene-1,3,6-trisulfonic acid, which renders the glycan fluorescent and provides a -3 charge for electrophoresis and negative-mode MS detection. The neutralization reaction also stabilizes the labile sialic acids. In addition to methylamidation, native charges from sialic acids were neutralized through reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium to amidate α2,6-linked sialic acids in the presence of ammonium chloride and form lactones with α2,3-linked sialic acids. This neutralization effectively labels each type of sialic acid with a unique mass to determine specific linkages on sialylated N-glycans. For both neutralization schemes, we compared the results from microchip electrophoresis and CE., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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