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Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis

✍ Scribed by Antonius Koller; Julia Khandurina; Jincai Li; Joel Kreps; David Schieltz; András Guttman

Publisher
John Wiley and Sons
Year
2004
Tongue
English
Weight
149 KB
Volume
25
Category
Article
ISSN
0173-0835

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✦ Synopsis

Abstract

We report on microbore liquid chromatography (μLC) and capillary electrophoresis (CE) separation of glycopeptides and high‐mannose‐type oligosaccharides, digested from recombinant phospholipase C, expressed in Pichia pastoris. The glycopeptides were subject to μLC/electrospray ionization/mass spectrometry (ESI‐MS) and μLC/ESI‐tandem MS (MS/MS) analysis that revealed high‐mannose structure size variation between Man~7~GlcNAc~2~ and Man~14~GlcNAc~2~. Then, high‐performance CE was applied to identify possible positional isomers of the high‐mannose structures. For the CE experiments, the oligosaccharides were released from the glycoproteins by peptide‐N‐glycosidase F and labeled with 1‐aminopyrene‐3,6,8‐trisulfonic acid (APTS). Excellent separation of the possible positional isomers was attained, suggesting one for Man~9~GlcNAc~2~, two for Man~10~GlcNAc~2~, three for Man~11~GlcNAc~2~, Man~12~GlcNAc~2~, and Man~13~GlcNAc~2~, and two for Man~14~GlcNAc~2~. The CE results provided complementary information to the μLC/ESI‐MS and MS/MS data with respect to the possible number of positional isomers.

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