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 Man9-GlcNAc2, two for Man10GlcNAc2, three for Man11GlcNAc2, Man12GlcNAc2, and Man13-GlcNAc2, 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.
- Capillary electrophoresis
- Microbore liquid chromatography-mass spectrometry
ASJC Scopus subject areas
- Analytical Chemistry
- Clinical Biochemistry