Engel Nicole Y, Weiss Victor U, Wenz Christian, Glück Susanne, Rüfer Andreas, Kratzmeier Martin, Marchetti-Deschmann Martina, Allmaier Günter
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164-IAC, 1060, Vienna, Austria.
Agilent Technologies, Hewlett-Packard-Straße 8, 76337, Waldbronn, Germany.
Anal Bioanal Chem. 2017 Nov;409(28):6625-6634. doi: 10.1007/s00216-017-0615-0. Epub 2017 Sep 20.
Due to the constant search for reliable methods to investigate glycoproteins in complex biological samples, an alternative approach combining affinity enrichment with rapid and sensitive analysis on-a-chip is presented. Glycoproteins were specifically captured by lectin-coated magnetic beads, eluted by competitive sugars, and investigated with microchip capillary gel electrophoresis (MCGE), i.e., CGE-on-a-chip. We compared our results to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data, which turned out to be in very good agreement. While SDS-PAGE offers the possibility of subsequent mass spectrometric analysis of captured and separated analytes, MCGE scores with time savings, higher throughput, and lower sample consumption as well as quality control (QC) and process analytical technology (PAT) applicability. Due to these advantages, a lectin-based glycoprotein capture protocol can easily be optimized. In our case, two different types of magnetic beads were tested and compared regarding lectin binding. The selectivity of our strategy was demonstrated with a set of model glycoproteins, as well as with human serum and serum depleted from high-abundance proteins. The specificity of the capturing method was investigated revealing to a certain degree an unspecific binding between each sample and the beads themselves, which has to be considered for any specific enrichment and data interpretation. In addition, two glycoproteins from Trichoderma atroviride, a fungus with mycoparasitic activity and only barely studied glycoproteome, were enriched by means of a lectin and so identified for the first time. Graphical abstract Glycoproteins from biological samples were detected by microchip capillary gel electrophoresis after lectin affinity enrichment using magnetic beads and elution with respective competitive monosaccharides.
由于一直在寻找可靠的方法来研究复杂生物样品中的糖蛋白,本文提出了一种将亲和富集与芯片上的快速灵敏分析相结合的替代方法。糖蛋白通过凝集素包被的磁珠特异性捕获,用竞争性糖类洗脱,并用微芯片毛细管凝胶电泳(MCGE)进行研究,即芯片上的CGE。我们将结果与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)数据进行了比较,结果显示非常吻合。虽然SDS-PAGE能够对捕获和分离的分析物进行后续质谱分析,但MCGE具有节省时间、高通量、低样品消耗以及质量控制(QC)和过程分析技术(PAT)适用性等优点。由于这些优势,基于凝集素的糖蛋白捕获方案可以很容易地进行优化。在我们的案例中,测试并比较了两种不同类型的磁珠在凝集素结合方面的情况。我们的策略的选择性通过一组模型糖蛋白以及人血清和去除高丰度蛋白的血清得到了证明。研究了捕获方法的特异性,结果在一定程度上揭示了每个样品与磁珠本身之间存在非特异性结合,这在任何特异性富集和数据解释中都必须加以考虑。此外,通过凝集素富集了来自绿色木霉的两种糖蛋白,绿色木霉是一种具有真菌寄生活性且糖蛋白组研究极少的真菌,这两种糖蛋白也是首次被鉴定出来。图形摘要 使用磁珠进行凝集素亲和富集并分别用竞争性单糖洗脱后,通过微芯片毛细管凝胶电泳检测生物样品中的糖蛋白。