Use of a porous graphitised carbon column for the high-performance liquid chromatography of oligosaccharides, alditols and glycopeptides with subsequent mass spectrometry analysis.
HPLC using a porous graphitised carbon (PGC) column eluted in acetonitrile-aqueous trifluoroacetic acid has been shown to give complementary chromatography to reversed-phase (ODS) HPLC for separation of peptides and glycopeptides. The PGC column can also be used for separation of oligosaccharides and oligosaccharide alditols released from protein by enzymes (N-linked chains) or base-borohydride degradation (O-linked chains). The advantages are that peptides, glycopeptides, reducing oligosaccharides, sialylated oligosaccharides and oligosaccharide alditols can be chromatographed under the same conditions. The samples can be readily recovered by evaporation for sensitive liquid secondary ion mass spectrometric (LSI-MS) analysis and there is no contamination or deterioration of chromatography from column leakage. LSI-MS analysis revealed that complete peak separation of all of the possible oligosaccharide components of the standard glycoproteins fetuin and bovine submaxillary mucin was not achieved. However, PGC remains as a useful adjunct to other HPLC profiling and separation techniques in particular where subsequent MS analysis is desired.
Davies
M J
MJ
Glycoconjugates Section, Clinical Research Centre, Watford Road, Harrow, UK.
Smith
K D
KD
Carruthers
R A
RA
Chai
W
W
Lawson
A M
AM
Hounsell
E F
EF
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006020:Glycopeptides / Q000302:isolation & purification*; D006108:Graphite; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification*; D012794:Sialic Acids / Q000737:chemistry; D013402:Sugar Alcohols / Q000302:isolation & purification*
D006020:Glycopeptides; D009844:Oligosaccharides; D012794:Sialic Acids; D013402:Sugar Alcohols; D006108:Graphite