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dc.contributor.authorBousfield, George R.en_US
dc.contributor.authorBaker, Vanda L.en_US
dc.contributor.authorGotschall, R. Russellen_US
dc.contributor.authorButnev, Viktor Y.en_US
dc.identifier.citationMethods (San Diego, Calif.). 2000 May; 21(1): 15-39.en_US
dc.descriptionClick on the DOI link below to access the article (may not be free).en_US
dc.description.abstractComplete carbohydrate composition analysis of glycoprotein hormones, their subunits, and oligosaccharides isolated from individual glycosylation sites can be accomplished using high-pH anion-exchange chromatography combined with pulsed amperometric detection. Neutral and amino sugars are analyzed from the same hydrolyzate by isocratic chromatography on a Dionex CarboPAC PA1 column in 16 mM NaOH. Sialic acid is quantified following mild hydrolysis conditions on the same column in 150 mM sodium acetate in 150 mM NaOH. Ion chromatography on a Dionex AS4A column in 1.8 mM Na(2)CO(3)/1.7 mM NaHCO(3); postcolumn, in-line anion micromembrane suppression; and conductivity detection can be used to quantify sulfate, a common component of pituitary glycoprotein hormone oligosaccharides. Mass spectrometric analysis before and after elimination of oligosaccharides from a single glycosylation site can provide an estimate of the average oligosaccharide mass, which facilitates interpretation of oligosaccharide composition data. Following release by peptide N-glycanase (PNGase) digestion and purification by ultrafiltration, oligosaccharides can be characterized by a high-resolution oligosaccharide mapping technique using the same equipment employed for composition analysis. Oligosaccharide mapping can be applied to the entire hormone, individual subunits, or individual glycosylation sites by varying PNGase digestion conditions or substrates. Oligosaccharide release by PNGase is readily monitored by SDS-PAGE. Site-specific deglycosylation can be confirmed by amino acid sequence analysis. For routine isolation of oligosaccharides, addition of 2-aminobenzamide at the reducing terminus facilitates detection; however, the oligosaccharide retention times are altered. Composition analysis is also affected as the 2-aminobenzamide-modified GlcNAc peak overlaps the fucose peak.en_US
dc.publisherAcademic Pressen_US
dc.relation.ispartofseriesMethods (San Diego, Calif.)en_US
dc.subject.meshAnthranilic Acids/chemistryen_US
dc.subject.meshChromatography, Ion Exchange/methodsen_US
dc.subject.meshGlycoprotein Hormones, alpha Subunit/chemistryen_US
dc.subject.meshGonadotropins, Equine/chemistryen_US
dc.subject.meshHydrogen-Ion Concentrationen_US
dc.subject.meshN-Acetylneuraminic Acid/chemistryen_US
dc.subject.meshPeptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidaseen_US
dc.subject.meshSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionizationen_US
dc.subject.meshTime Factorsen_US
dc.subject.meshGlycoprotein Hormones, alpha Subunit/isolation & purificationen_US
dc.titleCarbohydrate analysis of glycoprotein hormonesen_US
dc.description.versionpeer revieweden_US
dc.rights.holderCopyright 2000 Academic Press.en_US

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