Solid-phase oligosaccharide tagging (SPOT): Validation on glycolipid-derived structures
Solid-phase oligosaccharide tagging (SPOT): Validation on glycolipid-derived structures
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DOI：
10.1002/anie.200600642
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Hindsgaul, Ole
中科院分区：
文献类型：
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作者：
Lohse, Anders;Martins, Rita;Hindsgaul, Ole
糖基化是蛋白质翻译后修饰最常见的形式。在真核生物系统中，超过50%的蛋白质序列是糖基化的糖基化脂也广泛存在于动物细胞中：它们占细胞膜含量的5-10%，并导致一系列病理疾病糖蛋白和糖脂的低聚糖链的结构分析或测序比DNA或蛋白质的结构分析要复杂得多，因为分子通常是支链的，糖残基之间的连接引入了一个新的α或β立体中心。主要的分析方法是通过质谱（MS）或脉冲安培法检测低聚糖。通过化学或酶的方法从糖蛋白或糖脂中释放的低聚糖也经常在还原端进行标记，以提高检测的灵敏度通常的做法是插入荧光标记，然后进行色谱或电泳分析。然而，上述方法仍然是复杂的努力，需要复杂的设备和样品处理和数据分析方面的高度专业知识的结合。任何技术都有明显的优势，可以从溶液中分离出还原性低聚糖，并允许其进一步操作（包括标记），同时固定在固相上，例如，在珠或玻璃表面，如载玻片上。这就是固相低聚糖标记（SPOT）的基本原理，它只涉及移液和洗涤。优点是：•一旦低聚糖被“捕获”，它仍然与珠子共价结合，因此在随后的操作中不会有样品损失。•大量过量的标记（或其他）试剂可用于驱动反应完成，因为它们将被冲走而不会丢失或稀释样品。•如果固定的低聚糖仍然可以被酶和凝集素（或抗体）立体接近，生物低聚糖识别和酶辅助
Glycosylation is the most abundant form of post-translational modification of proteins. Over 50% of all protein sequences are glycosylated in eukaryotic systems.[1] Glycosylated lipids are also widespread in animal cells: they constitute up to 5–10% of the cell membrane content and are responsible for a wide array of pathological disorders.[2] The structural analysis, or sequencing, of the oligosaccharide chains of glycoproteins and glycolipids is much more complex than that of DNA or proteins, as the molecules are generally branched and the linkage between sugar residues introduces a new α or β stereocenter. The major analytical approaches [3] rely on the detection of oligosaccharides either by mass spectrometry (MS) or pulsed amperometry. Oligosaccharides released from glycoproteins or glycolipids by chemical or enzymatic methods are also frequently labeled at the reducing end to afford increased sensitivity of detection.[4] The insertion of fluorescent tags followed by analysis by chromatography or electrophoresis is common practice. The above approaches, however, remain complex endeavors that require a combination of sophisticated equipment and a high degree of expertise in both sample handling and data analysis. There would be clear advantages in any technique that could sequester a reducing oligosaccharide from solution and allow its further manipulation (including tagging) while immobilized on a solid phase, for example, on beads or glass surfaces such as slides. This is the rationale behind solidphase oligosaccharide tagging (SPOT) that would involve only pipetting and washing. The advantages are:• Once the oligosaccharide is “captured” it remains covalently bound to the beads, so there would be no sample loss during subsequent manipulations.• Large excesses of tagging (or other) reagents could be used to drive reactions to completion, as they would be washed away without sample loss or dilution.• If the immobilized oligosaccharide remained sterically accessible to enzymes and lectins (or antibodies), biological oligosaccharide recognition and enzyme-assisted