High-Throughput Analysis of the Plasma N-Glycome by UHPLC
High-Throughput Analysis of the Plasma N-Glycome by UHPLC
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DOI：
10.1007/978-1-4939-6493-2_8
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发表时间：
2017-01-01
期刊：
影响因子：
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通讯作者：
Rudd, Pauline M.
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文献类型：
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作者：
Adamczyk, Barbara;Stockmann, Henning;Rudd, Pauline M.
对健康和疾病中糖基化改变的理解已经显着发展，聚糖被认为是相关的候选生物标志物。能够生成高质量、大规模糖谱分析数据的高通量分析技术的需求量很大。在这里，我们描述了自动化样品制备工作流程，以及使用亲水相互作用液相色谱和超高效液相色谱 (UHPLC) 仪器上的荧光检测对血浆样品中的 N 连接聚糖进行分析。样品在 96 孔板中制备，工作流程包括快速糖蛋白变性、酶促聚糖释放、固相酰肼上的聚糖纯化、荧光标记和固相萃取标记后净化。血浆 N-聚糖分析新方法的开发及其在机器人平台上的实施显着减少了样品制备所需的时间，并最大限度地减少了技术变化。预计所开发的方法将有助于扩展分析蛋白质糖基化的高通量能力。
The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofi ling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation.