HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation

被引:31
作者
He, ZC
Aristoteli, LP
Kritharides, L
Garner, B [1 ]
机构
[1] Univ New S Wales, Sch Med Sci, Sydney, NSW 2052, Australia
[2] Heart Res Inst, Camperdown, NSW 2050, Australia
[3] Univ New S Wales, Fac Med, Ctr Vasc Res, Sydney, NSW 2052, Australia
[4] Univ Sydney, Concord Hosp, Dept Cardiol, Sydney, NSW 2006, Australia
[5] Prince Wales Med Res Inst, Randwick, NSW 2031, Australia
基金
澳大利亚研究理事会;
关键词
N-glycan analysis; haptoglobin; glycomics; proteomics; disease markers;
D O I
10.1016/j.bbrc.2006.03.007
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (c) 2006 Elsevier Inc. All rights reserved.
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页码:496 / 503
页数:8
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