Effect of glycosylation on the protein pattern in 2-D-gel electrophoresis
Effect of glycosylation on the protein pattern in 2-D-gel electrophoresis
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DOI：
10.1002/pmic.200600297
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发表时间：
2007-01-01
期刊：
影响因子：
3.4
通讯作者：
Troxler, Heinz
中科院分区：
文献类型：
--
作者：
Kleinert, Peter;Kuster, Thomas;Troxler, Heinz
当用2-D-PAGE分析单个蛋白质时，由于PTM，通常显示多个斑点。在人体体液的凝胶中，斑点模式有助于蛋白质的分配和鉴定。我们分析了先天性糖基化障碍（CDG）患者的血清，其中糖蛋白受到强烈影响，与健康对照组相比，显示出高度可区分的斑点模式。我们检测到α（1）-酸性糖蛋白（AGP）和转铁蛋白（Trf）的典型蛋白质模式，这是CDG的标志物。AGP含有五个糖基化位点，这导致糖蛋白的复杂微观异质性。另一方面，在Trf中，结合的是仅具有两个糖基化位点的糖蛋白，主要是双触角复合型N-连接聚糖。我们使用2-D-PAGE、MALDI-TOF-MS和ESI-MS对这些糖蛋白及其相应的聚糖进行分析。在AGP中，不同糖基化位点的异源糖基化是造成复杂斑点模式的原因。与AGP相反，Trf的蛋白质斑点不能用糖基化来解释。我们发现了强有力的证据，半胱氨酸的氧化是负责的斑点图案。这项研究反驳了普遍接受的假设，即在2-D-PAGE中观察到的多个蛋白质点的Trf是由于，如在AGP中，糖基化的蛋白质。
Single proteins, when analyzed with 2-D-PAGE, often show multiple spots due to PTMs. In gels of human body fluids, the spot patterns facilitate the assignment and identification of the proteins. We analyzed serums from patients with congenital disorders of glycosylation (CDG) in which glycoproteins are strongly impacted and exhibit highly distinguishable spot patterns compared to healthy controls. We detected a typical protein pattern for alpha(1)-acid glycoprotein (AGP) and transferrin (Trf) that are markers for CDG. AGP contains five glycosylation sites which results in a complex microheterogeneity of the glycoprotein. on the other hand, in Trf, a glycoprotein with only two glycosylation sites, mainly biantennary complex-type-N-linked glycans are bound. We used 2-D-PAGE, MALDI-TOF-MS, and ESI-MS for the analysis of these glycoproteins and their corresponding glycans. In AGP, the heterogenic glycosylation of the different glycosylation sites is responsible for the complex spot pattern. In contrast to AGP, the protein spots of Trf cannot be explained by glycosylation. We found strong evidence that oxidation of cysteine is responsible for the spot pattern. This study contradicts the commonly accepted assumption that the multiple protein spots of Trf observed in 2-D-PAGE are due, as in AGP, to the glycosylation of the protein.