Enhanced detection of sialylated and sulfated glycans with negative ion mode nanoliquid chromatography/mass spectrometry at high pH.
Negative ion mode nanoliquid chromatography/mass spectrometry (nano-LC/MS) on porous graphitic carbon columns at pH 11 was studied and compared to capillary LC/MS at pH 8 for the analysis of neutral and acidic glycan alditols. Oligosaccharides were chromatographed with an acetonitrile gradient containing 0.04% ammonium hydroxide and analyzed with a linear ion trap mass spectrometer (LTQ) equipped with a modified nanospray interface. Analysis of acidic N- and O-glycan standards revealed that good quality MS/MS spectra could be obtained when loading 1-3 fmol, a 10-fold increase in sensitivity compared to capillary-LC/MS at pH 8. Analysis of a complex mixture of O-glycans from porcine colonic mucins with nano-LC/MS and MS/MS at high pH revealed 170 oligosaccharides in one analysis, predominantly corresponding to sulfated glycans with up to 11 residues. Analysis of the same sample with capillary-LC/MS showed a lower sensitivity for multiply sulfated glycans. Nano-LC/MS of O-linked oligosaccharides on MUC2 from a human colon biopsy also illustrated that the ionization of oligosaccharides with multiple sialic acid groups was increased compared to those with only one sialic acid residue. Nano-LC/MS at high pH is, thus, a highly sensitive approach for the analysis of acidic oligosaccharides.
Thomsson
Kristina A
KA
Department of Medical Biochemistry, University of Gothenburg, Box 440, 405 30 Gothenburg, Sweden. kristina.thomsson@medkem.gu.se
Bäckström
Malin
M
Holmén Larsson
Jessica M
JM
Hansson
Gunnar C
GC
Karlsson
Hasse
H
D000818:Animals; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D036103:Nanotechnology; D009584:Nitrogen / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry*; D012015:Reference Standards; D013464:Sulfuric Acids / Q000737:chemistry*
D009077:Mucins; D009844:Oligosaccharides; D011134:Polysaccharides; D013464:Sulfuric Acids; D019158:N-Acetylneuraminic Acid; D009584:Nitrogen; C033158:sulfuric acid; D010100:Oxygen