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

• Published in: Electrophoresis Vol. 25; no. 13; pp. 2003 - 2009
• Main Authors: Koller, Antonius, Khandurina, Julia, Li, Jincai, Kreps, Joel, Schieltz, David, Guttman, András
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.07.2004; WILEY‐VCH Verlag
• Subjects: Capillary electrophoresis; Chromatography, Liquid - methods; Electrophoresis, Capillary - methods; Glycopeptides; Glycopeptides - analysis; Glycopeptides - chemistry; glycoproteins; ionization; Isomerism; Komagataella pastoris; liquid chromatography; Mannose - chemistry; Mass Spectrometry - methods; Microbore liquid chromatography-mass spectrometry; Microchemistry; Oligosaccharides; Oligosaccharides - analysis; Oligosaccharides - chemistry; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism; phospholipase C; Pichia - genetics; Pichia - metabolism; Pyrenes - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Time Factors; Type C Phospholipases - genetics; Type C Phospholipases - metabolism
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.200305837

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 Man7GlcNAc2 and Man14GlcNAc2. 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 Man9GlcNAc2, two for Man10GlcNAc2, three for Man11GlcNAc2, Man12GlcNAc2, and Man13GlcNAc2, and two for Man14GlcNAc2. The CE results provided complementary information to the μLC/ESI‐MS and MS/MS data with respect to the possible number of positional isomers.