Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification
Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification
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DOI：
10.1074/mcp.m800308-mcp200
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发表时间：
2009-01-01
影响因子：
7
通讯作者：
Hirabayashi, Jun
中科院分区：
文献类型：
--
作者：
Kuno, Atsushi;Kato, Yukinari;Hirabayashi, Jun
蛋白质糖基化是蛋白质组学领域日益受到关注的一个关键课题，因为它有望在组织学和诊断生物标志物的研究中发挥关键作用。在这种背景下，大量糖蛋白现已被指定为疾病相关生物标志物。然而，当前的蛋白质组平台中没有适当的策略来鉴定此类标记候选分子，从而将它们的特异性表达与特定疾病联系起来。在这里，我们提出了一种新的实用系统，用于在抗体辅助凝集素分析（ALP）方面进行集中差异聚糖分析。在开发的程序中，（i）通过使用识别核心蛋白部分的特异性抗体进行免疫沉淀，从临床样品（例如组织提取物、细胞上清液或血清）中富集靶蛋白； (ii) 使用与 (i) 中使用的相同抗体通过免疫印迹对靶糖蛋白进行定量； (iii)通过抗体覆盖凝集素微阵列分析糖基化差异，这是一种新兴的聚糖分析微阵列的应用技术。作为具有N-连接或O-连接聚糖的模型糖蛋白，分别对前列腺特异性抗原或平足蛋白进行系统的ALP分析。结果，借助特异性抗体，在亚皮摩尔水平上获得了与目标糖蛋白聚糖相对应的特异性信号，从而可以以快速、可重复和高通量的方式观察疾病特异性或组织特异性糖基化变化。因此，已建立的系统应该提供强大的管道来支持糖生物标志物发现的持续努力。分子与细胞蛋白质组学 8:99-108, 2009。
Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e. g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of ongoing efforts in glyco-biomarker discovery. Molecular & Cellular Proteomics 8:99-108, 2009.