Szigeti Marton, Guttman Andras
Horvath Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.
MTA-PE Translational Glycomics Group, University of Pannonia, H-8200 Veszprem, Egyetem u 10, Veszprem, Hungary.
Methods Mol Biol. 2017;1503:265-272. doi: 10.1007/978-1-4939-6493-2_19.
N-glycan profiling of therapeutic glycoproteins is essential to ensure the activity and efficacy of these promising new-generation drugs. The N-linked glycan moieties of these entities highly affect circulation half-life, immunogenicity and receptor-binding activity as well as physicochemical and thermal stability properties. In addition, more than half of the biopharmaceuticals are glycoproteins representing multibillion dollar worldwide business, further emphasizing the importance of their analysis. In the biomedical field, on the other hand, revealing disease-related glycan structure alterations holds the promise of the discovery of new biomarkers for early diagnostics. Therefore, there is a great demand for widely applicable, high-throughput sample preparation and analysis methods for N-glycan profiling of glycoproteins. One of the newest exciting developments of the field is the magnetic bead based glycoprotein sample preparation technique. A detailed protocol of this method is given in this chapter in conjunction with rapid capillary electrophoresis analysis of the prepared samples by laser induced fluorescence detection (CE-LIF). N-glycans are digested by the endoglycosidase PNGase F and the released carbohydrates are labeled with the charged fluorophore dye of aminopyrenetrisulfonate (APTS). Effective glycan capture by magnetic microparticles enabled fast, easily automated sample preparation both in individual (single vial) and 96-well plate formats, including excess dye removal. Rapid separation of APTS labeled IgG glycans is also shown utilizing an optimized CE-LIF protocol.
治疗性糖蛋白的N-聚糖分析对于确保这些有前景的新一代药物的活性和疗效至关重要。这些物质的N-连接聚糖部分对循环半衰期、免疫原性和受体结合活性以及物理化学和热稳定性特性有很大影响。此外，超过一半的生物制药产品是糖蛋白，代表着全球数十亿美元的业务，这进一步凸显了对其进行分析的重要性。另一方面，在生物医学领域，揭示与疾病相关的聚糖结构改变有望发现用于早期诊断的新生物标志物。因此，对于广泛适用的、高通量的糖蛋白N-聚糖分析样品制备和分析方法有很大需求。该领域最新的一项令人兴奋的进展是基于磁珠的糖蛋白样品制备技术。本章结合通过激光诱导荧光检测（CE-LIF）对制备好的样品进行快速毛细管电泳分析，给出了该方法的详细方案。N-聚糖被内切糖苷酶PNGase F消化，释放出的碳水化合物用氨基芘三磺酸盐（APTS）的带电荷荧光团染料进行标记。磁性微粒对聚糖的有效捕获使得能够以单个（单瓶）和96孔板形式快速、轻松地实现自动化样品制备，包括去除过量染料。利用优化的CE-LIF方案还展示了对APTS标记的IgG聚糖的快速分离。
