Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry
Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry
复制标题
DOI：
10.1074/mcp.m400068-mcp200
复制
发表时间：
2005-02-01
影响因子：
7
通讯作者：
Roepstorff, P
中科院分区：
文献类型：
--
作者：
Larsen, MR;Hojrup, P;Roepstorff, P
蛋白质糖基化对于改变蛋白质的功能或理化性质至关重要。糖基化异常可导致蛋白质功能失常，从而导致严重的疾病。因此，开发与最先进的蛋白质组学相当的灵敏度水平的蛋白质修饰表征技术是很重要的。虽然存在表征高丰度糖蛋白的技术，但目前没有一种方法能够同时提供从电泳凝胶中切除的单个条带上的位点占用和聚糖结构的信息。我们提出了一种新的技术，允许表征低量的糖蛋白分离凝胶电泳。该方法采用顺序特异性和非特异性酶处理，然后使用石墨粉微柱结合质谱法对糖肽进行选择性纯化和表征。该方法比以前的方法更快，更敏感，并且与蛋白质组学研究兼容。
Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.