Carbohydrate analysis of glycoprotein hormones

Complete 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 w ith pulsed amperometric detection. Neutral and amino sugars are analyzed fr om the same hydrolyzate by isocratic chromatography on a Dionex CarboPAC PA 1 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 Na2CO3/1.7 mM NaHCO3; pos tcolumn, in-line anion micromembrane suppression; and conductivity detectio n can be used to quantify sulfate, a common component of pituitary glycopro tein hormone oligosaccharides. Mass spectrometric analysis before and after elimination of oligosaccharides from a single glycosylation site can provi de an estimate of the average oligosaccharide mass, which facilitates inter pretation of oligosaccharide composition data. Following release by peptide N-glycanase (PNGase) digestion and purification by ultrafiltration, oligos accharides can be characterized by a high-resolution oligosaccharide mappin g technique using the same equipment employed for composition analysis. Oli gosaccharide mapping can be applied to the entire hormone, individual subun its, or individual glycosylation sites by varying PNGase digestion conditio ns or substrates. Oligosaccharide release by PNGase is readily monitored by SDS-PAGE. Site-specific deglycosylation can be confirmed by amino acid seq uence analysis. For routine isolation of oligosaccharides, addition of 2-am inobenzamide at the reducing terminus facilitates detection; however, the o ligosaccharide retention times are altered. Composition analysis is also af fected as the 2-aminobenzamide-modified GlcNAc peak overlaps the fucose pea k. (C) 2000 Academic Press.