The University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Australia.
J Proteome Res. 2010 Oct 1;9(10):5496-500. doi: 10.1021/pr100472z.
Alterations in protein glycosylation play an important role in patho-physiology, and much effort has been devoted to detecting glycoprotein biomarkers. In this manuscript, we describe the development of a novel method for monitoring alterations in protein glycosylation. Lectins are used as individual affinity reagents and coupled to magnetic beads (Dynabeads) in a microplate array format for isolation of glycosylated proteins. Isolated glycoproteins are digested with trypsin in-solution followed by LC-MS/MS, allowing a liquid handler-assisted high throughput workflow. We demonstrate the specific and reproducible affinity-isolation of glycoproteins using the lectin Dynabead array technology. When used with serum, we achieved one-step purification of glycoproteins with minimal coisolation of abundant serum proteins including albumin. We further optimized the proteomics workflow to allow transfer to a liquid handler for automation. In summary, we report the development of a high throughput platform to detect alterations in protein glycosylation which will be useful in glycoproteomics studies, particularly clinical proteomics studies where large sample sizes are required to achieve statistical power.
蛋白质糖基化的改变在病理生理学中起着重要作用，人们已经投入了大量的精力来检测糖蛋白生物标志物。在本文中，我们描述了一种用于监测蛋白质糖基化改变的新方法的开发。凝集素被用作单个亲和试剂，并以微板阵列的形式偶联到磁珠（Dynabeads）上，用于分离糖基化蛋白质。分离的糖蛋白在溶液中用胰蛋白酶消化，然后进行 LC-MS/MS，允许进行液体处理辅助的高通量工作流程。我们使用凝集素 Dynabead 阵列技术证明了糖蛋白的特异性和可重现性亲和分离。当用于血清时，我们实现了一步法糖蛋白纯化，同时最小化了包括白蛋白在内的丰富血清蛋白的共分离。我们进一步优化了蛋白质组学工作流程，以允许转移到液体处理机进行自动化。总之，我们报告了一种高通量平台的开发，用于检测蛋白质糖基化的改变，这将在糖蛋白质组学研究中非常有用，特别是在需要大量样本量才能达到统计效力的临床蛋白质组学研究中。
