Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis.
We report on microbore liquid chromatography (microLC) 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 microLC/electrospray ionization/mass spectrometry (ESI-MS) and microLC/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 microLC/ESI-MS and MS/MS data with respect to the possible number of positional isomers.
Koller
Antonius
A
Diversa Corporation, San Diego, CA, USA.
Khandurina
Julia
J
Diversa Corporation, San Diego, CA, USA.
Li
Jincai
J
Diversa Corporation, San Diego, CA, USA.
Kreps
Joel
J
Diversa Corporation, San Diego, CA, USA.
Schieltz
David
D
Diversa Corporation, San Diego, CA, USA.
Guttman
András
A
Diversa Corporation, San Diego, CA, USA.
D002853:Chromatography, Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry; D007536:Isomerism; D008358:Mannose / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008832:Microchemistry; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011721:Pyrenes / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism
C098210:1-aminopyrene-3,6,8-trisulfonic acid; D006020:Glycopeptides; D009844:Oligosaccharides; D011721:Pyrenes; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose