Structural analysis of N- and O-glycans released from glycoproteins
Structural analysis of N- and O-glycans released from glycoproteins
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DOI：
10.1038/nprot.2012.063
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发表时间：
2012-07-01
期刊：
影响因子：
14.8
通讯作者：
Packer, Nicolle H.
中科院分区：
文献类型：
--
作者：
Jensen, Pia H.;Karlsson, Niclas G.;Packer, Nicolle H.
该方案显示了如何从纯化的糖蛋白或蛋白质混合物中获得详细的聚糖组成和结构谱，并且它可用于区分不同的同量异序聚糖异构体。将糖蛋白固定在PVDF膜上，然后通过PNGase F酶促释放N-聚糖，分离并还原。随后，O-聚糖通过还原性β-消除从相同的蛋白质点化学释放。经阳离子交换微柱净化后，用多孔石墨化碳液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）进行分离和分析。任选地，可以用唾液酸酶或其他特异性外切糖苷酶处理聚糖以产生更详细的结构信息。样品制备大约需要4天，第2天和第3天的工作量较大，第1天和第4天的工作量较小。数据解释的时间取决于所分析样品的复杂性。该方法可与通过毛细管/nanoLC-ESI-MS/MS分析富集的糖肽结合使用，共同提供有关糖基化位点异质性的详细信息。
This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive beta-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.