Sparbier Katrin, Koch Sonja, Kessler Irina, Wenzel Thomas, Kostrzewa Markus
Bruker Daltonik GmbH, Leipzig, Germany.
J Biomol Tech. 2005 Dec;16(4):407-13.
Glycosylation is the most common form of posttranslational modification of proteins (50-80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid-functionalized beads.
糖基化是蛋白质翻译后修饰最常见的形式（占50 - 80%）。糖基化肽和蛋白质的分离、发现及后续鉴定在糖蛋白质组学和诊断中变得越来越重要。基质辅助激光解吸电离飞行时间质谱（MALDI - TOF质谱）是鉴定肽和蛋白质及其相应修饰的理想技术。通过功能化磁性颗粒支持的亲和色谱法，可以从不同来源富集糖基化肽和蛋白质。分别采用碳二亚胺法和聚戊二醛法将伴刀豆球蛋白A（ConA）和二硼酸共价包被在磁珠上。利用功能化磁珠，针对自动化工作流程以及随后通过MALDI - TOF质谱进行的检测和鉴定，建立并优化了糖基化肽和蛋白质的结合与检测方案。对于几种模型蛋白质，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和MALDI - TOF质谱证明了其捕获和鉴定效果。根据糖基化类型（高甘露糖型、杂合型或复合型），不同的蛋白质通过ConA或硼酸功能化磁珠进行富集。
