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

We report on microbore liquid chromatography (muLC) 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 muLC/electrospray ionization/mass spectrometry (ESI-MS) and muC/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 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 muLC/ESI-MS and MS/MS data with respect to the possible number of positional isomers.