Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis.
A new beta-elimination-based procedure has been devised for a microscale release of O-linked oligosaccharides from glycoproteins. Unlike the conventional Carlson degradation, which leads to formation of alditols, the procedure reported here renders the reducing end intact. Conversion of the liberated oligosaccharides to glycosylamines in ammonia medium is followed by the production of the reducing oligosaccharides through the addition of boric acid. The quantitatively generated oligosaccharides with the reducing end can subsequently be derivatized with a fluorophoric reagent for capillary electrophoresis or, alternatively, analyzed through MALDI mass spectrometry. The microscale version of these chemical steps permits us to investigate structurally O-linked oligosaccharides at very low levels.
Huang
Y
Y
Department of Chemistry, Indiana University, Bloomington 47405, USA.
Mechref
Y
Y
Novotny
M V
MV
D000641:Ammonia / Q000737:chemistry; D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary; D005936:Glucans / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D007202:Indicators and Reagents; D008895:Milk, Human / Q000737:chemistry; D009077:Mucins / Q000032:analysis; D036103:Nanotechnology; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification; D011721:Pyrenes; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D002787:Sterol Esterase / Q000032:analysis; D000509:alpha-Fetoproteins / Q000737:chemistry; D062367:ortho-Aminobenzoates
C103430:8-aminopyrene-1,3,6-trisulfonic acid; D005936:Glucans; D006023:Glycoproteins; D007202:Indicators and Reagents; D009077:Mucins; D011134:Polysaccharides; D011721:Pyrenes; D000509:alpha-Fetoproteins; D062367:ortho-Aminobenzoates; C031146:maltoheptaose; D000641:Ammonia; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; C000219:anthranilamide