Isolation and purification of glycoconjugates from complex biological sources by recycling high-performance liquid chromatography.
Among of the most urgent needs of the glycobiology community is to generate libraries of pure carbohydrate standards. While many oligosaccharides have recently been synthesized, some glycans of biomedical importance are still missing in existing collections or are available in only limited amounts. To address this need, we demonstrate the use of the relatively unexplored technique of recycling high-performance liquid chromatography (R-HPLC) to isolate and purify glycoconjugates from several natural sources. We were able to routinely achieve purities greater than 98%. In several cases, we were able to obtain isomerically pure substances, particularly for glycans with different positional isomerism. These purified substances can then be used in different analytical applications, for example, as standards for mass spectrometry (MS) and capillary-based separations. Moreover, using a bifunctional aromatic amine, the same derivatization agent can be used to enable UV detection of oligosaccharides during their purification and link the isolated molecules to functionalized surfaces and potentially create glycan arrays.
Department of Chemistry Indiana University Bloomington, Indiana 47405, United States.
William R
WR
Alley
Benjamin F
BF
Mann
Vlastimil
V
Hruska
Milos V
MV
Novotny
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006001:Glycoconjugates / Q000302:isolation & purification*; D006801:Humans; D008895:Milk, Human / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D053719:Tandem Mass Spectrometry
D006001:Glycoconjugates