Bunkenborg Jakob, Pilch Bartosz J, Podtelejnikov Alexandre V, Wiśniewski Jacek R
MDS Proteomics Odense, Denmark.
Proteomics. 2004 Feb;4(2):454-65. doi: 10.1002/pmic.200300556.
In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.
在过去几年中，质谱分析法已成为表征翻译后修饰蛋白质的首选方法。大多数蛋白质化学修饰在某种意义上是二元的，即对于给定残基只能有一个变化，而许多不同的寡糖可以连接到糖基化位点残基上。在复杂生物样品中对糖蛋白进行详细表征极具挑战性。然而，可以在中间水平获得有关N-糖基化的信息。在此，我们展示了一种通过降低样品复杂性和富集糖蛋白含量来绘制复杂混合物中N-糖基化位点的方法。通过初始凝集素色谱步骤选择糖基化蛋白，并用内肽酶Lys-C进行消化。然后通过第二个凝集素色谱步骤从消化混合物中选择糖基化肽。在用N-糖苷酶F去除聚糖成分并用胰蛋白酶消化肽后，通过在线反相液相色谱质谱法进行分析。使用两种不同的凝集素，伴刀豆球蛋白A和麦胚凝集素，将该方法应用于人血清，共鉴定出77种蛋白质中的86个N-糖基化位点。
