Hinneburg Hannes, Schirmeister Falko, Korać Petra, Kolarich Daniel
Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424, Potsdam, Germany.
Department of Biology, Chemistry, Pharmacy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195, Berlin, Germany.
Methods Mol Biol. 2017;1503:131-145. doi: 10.1007/978-1-4939-6493-2_11.
The availability of well-defined samples in sufficient numbers represents a major bottleneck for any biomarker related research. The utilization of preserved, archived and clinically well-described samples therefore holds a great potential to bridge this gap. This chapter describes a universal workflow for the comprehensive characterization of N- and O-glycans released from whole formalin-fixed, paraffin-embedded tissue sections, including an option for further partitioning using laser microdissection of specific tissue areas/cell populations. Glycoproteins are extracted and subsequently immobilized onto a PVDF membrane prior enzymatic release of N-glycans. Following N-glycan retrieval O-glycans are released using reductive β-elimination from the same sample spot, significantly reducing the required amount of starting material. Released and reduced glycan structures are characterized using porous graphitized carbon liquid chromatography online coupled to an electrospray ionization-ion trap mass spectrometer. This technique provides information on the relative abundances of individual glycans along with detailed structural information, including isomer differentiation and functional epitope characterization of N- and O-glycans obtained from minimal amounts of tissue down to a few thousand cells.
能否获得数量充足、定义明确的样本是任何与生物标志物相关研究的主要瓶颈。因此，利用保存的、存档的以及临床描述详尽的样本极有可能弥补这一差距。本章介绍了一种通用工作流程，用于全面表征从福尔马林固定、石蜡包埋的全组织切片中释放的N-聚糖和O-聚糖，包括使用激光显微切割特定组织区域/细胞群体进行进一步分离的选项。在酶促释放N-聚糖之前，先提取糖蛋白并将其固定在聚偏二氟乙烯（PVDF）膜上。在回收N-聚糖后，使用还原β-消除法从同一样本点释放O-聚糖，从而显著减少起始材料的需求量。使用与电喷雾电离-离子阱质谱仪在线联用的多孔石墨化碳液相色谱法对释放并还原后的聚糖结构进行表征。该技术可提供有关各个聚糖相对丰度的信息以及详细的结构信息，包括从少量组织直至几千个细胞中获得的N-聚糖和O-聚糖的异构体区分和功能表位表征。
