Mass + Retention Time = Structure: A Strategy for the Analysis of N-Glycans by Carbon LC-ESI-MS and Its Application to Fibrin N-Glycans

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• 作者：Martin Pabst ; Jayakumar Singh Bondili ; Johannes Stadlmann ; Lukas Mach ; Friedrich Altmann
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：July 1, 2007
• 年：2007
• 卷：79
• 期：13
• 页码：5051 - 5057
• 全文大小：265K
• 年卷期：v.79,no.13(July 1, 2007)
• ISSN：1520-6882

文摘

Analysis of the numerous possible, often isobaric structures of protein-bound oligosaccharides calls for a high-performance two-dimensional method that combines liquid chromatography's ability to separate isomers andmass spectrometry's ability to determine glycan composition. Here we investigate the usefulness of porous graphitic carbon columns coupled to ESI-MS for the separation of N-glycans with two or more sialic acids. Internalstandards helped to rectify retention time fluctuations andthus allowed elution times to play an essential role in thestructural assignment of peaks. For generation of aretention time library, standards representing the possibleisomers of diantennary non-, mono-, and disialylatedN-glycans, differing in the linkage of galactose and sialicacids as well as isobaric hybrid-type N-glycans, wereproduced using recombinant glycosyltransferases. Oncethe retention times library was established, isomers couldbe identified by LC-ESI-MS in the positive mode withoutadditional MS/MS experiments. The method was appliedfor the detailed structural analysis of fibrin(ogen) N-glycans from various species (human, cow, pig, mouse,rat, cat, dog, Chinese hamster, horse, sheep, and chicken).All fibrins contained diantennary N-glycans. They differedin the occurrence of 1,3-linked galactose, 2,3-linkedsialic acids, and N-glycolylneuraminic acid, in the mono/diantennary glycan ratio, and in the O-acetylation ofneuraminic acids. The separation system's potential foranalyzing tri- and tetrasialylated N-glycans was demonstrated.