Bodnar Judit, Szekrenyes Akos, Szigeti Marton, Jarvas Gabor, Krenkova Jana, Foret Frantisek, Guttman Andras
MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem, Hungary.
Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.
Electrophoresis. 2016 May;37(10):1264-9. doi: 10.1002/elps.201500575. Epub 2016 Mar 15.
Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N-glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high-throughput, and reproducible methods to analyze protein N-glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N-glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme-coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in-solution enzyme reactions using standard glycoproteins possessing the major N-glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in-solution based method in 10-min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.
蛋白质糖基化研究是生物标志物发现和生物制药研究中的一个重要领域。蛋白质N-糖基化的改变可能表明医学领域病理状况的变化或生物治疗药物的生产参数变化。这些学科的快速发展需要快速、高通量且可重复的方法来分析蛋白质N-糖基化。目前使用的方法要么需要较长的去糖基化时间，要么需要大量过量的酶。在本文中，我们报道了将PNGase F固定在磁性微粒表面，并将其用于从糖蛋白中快速有效地去除N-聚糖。使用固定化的PNGase F还使得酶包被的珠子能够重复使用，因为在每次去糖基化反应后，可以通过磁铁轻松地将磁性微粒从样品中分离出来。将PNGase F包被的磁珠的效率和活性与使用具有中性、高甘露糖和高唾液酸化碳水化合物等主要N-聚糖类型的标准糖蛋白进行的溶液内酶反应进行了比较。对于免疫球蛋白G（主要是中性碳水化合物）、核糖核酸酶B（高甘露糖型糖）和胎球蛋白（高唾液酸化寡糖）等模型糖蛋白，PNGase F包被的磁珠在10分钟的反应时间内提供了与传统溶液法相当的去糖基化水平，具有易于从反应混合物中去除酶和可重复使用的特点。
