HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation.
Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples.
School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
Zhicong
Z
He
Lina P
LP
Aristoteli
Leonard
L
Kritharides
Brett
B
Garner
D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D006242:Haptoglobins / Q000032:analysis* / Q000737:chemistry*; D006801:Humans; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry*; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry
D006242:Haptoglobins; D009844:Oligosaccharides; D020033:Protein Isoforms