N-glycosylation site occupancy in serum glycoproteins using multiple reaction monitoring liquid chromatography-mass spectrometry
N-glycosylation site occupancy in serum glycoproteins using multiple reaction monitoring liquid chromatography-mass spectrometry
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DOI：
10.1074/mcp.m700361-mcp200
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发表时间：
2007-12-01
影响因子：
7
通讯作者：
Hennet, Thierry
中科院分区：
文献类型：
--
作者：
Huelsmeier, Andreas J.;Paesold-Burda, Patricie;Hennet, Thierry
先天性糖基化障碍（CDG）是一个与严重临床表现相关的N-连接糖基化缺陷家族。在CDG I型中，脂质连接的寡糖组装的缺陷导致糖蛋白上的N-糖基化位点的占用不足。尽管已知残余糖基化活性水平与与个体CDG突变相关的临床表型相关，但尚不清楚N-糖基化位点占用程度本身是否与疾病的严重程度相关。为了量化健康对照和CDG样品中糖基化不足的程度，我们开发了一种基于多反应监测LC-MS/MS的N-糖基化位点占有率的定量方法。使用同位素标记的标准肽，我们直接量化了选定血清蛋白的N-糖基化位点占有率水平。在健康对照样品中，我们测定了转铁蛋白和α（1）-抗胰蛋白酶的所有N-糖基化位点的98-100%占用率。在CDG I型样本中，我们观察到与疾病严重程度相关的N-糖基化位点占有率降低。此外，我们注意到N-糖基化位点的选择性糖基化不足，表明给定糖蛋白的受体序列的优先糖基化。在转铁蛋白中，观察到第一个N-糖基化位点的优先占据，而在α（1）-抗胰蛋白酶中观察到第一、第三和第二个N-糖基化位点的优先性降低。这种多反应监测LC-MS/MS方法可以扩展到多种糖蛋白，从而能够对生物样品中的N-糖基化位点进行糖蛋白质组学调查。
Congenital disorders of glycosylation (CDGs) are a family of N-linked glycosylation defects associated with severe clinical manifestations. In CDG type-I, deficiency of lipid-linked oligosaccharide assembly leads to the underoccupancy of N-glycosylation sites on glycoproteins. Although the level of residual glycosylation activity is known to correlate with the clinical phenotype linked to individual CDG mutations, it is not known whether the degree of N-glycosylation site occupancy by itself correlates with the severity of the disease. To quantify the extent of underglycosylation in healthy control and in CDG samples, we developed a quantitative method of N-glycosylation site occupancy based on multiple reaction monitoring LC-MS/MS. Using isotopically labeled standard peptides, we directly quantified the level of N-glycosylation site occupancy on selected serum proteins. In healthy control samples, we determined 98-100% occupancy for all N-glycosylation sites of transferrin and alpha(1)-antitrypsin. In CDG type-I samples, we observed a reduction in N-glycosylation site occupancy that correlated with the severity of the disease. In addition, we noticed a selective underglycosylation of N-glycosylation sites, indicating preferential glycosylation of acceptor sequons of a given glycoprotein. In transferrin, a preferred occupancy for the first N-glycosylation site was observed, and a decreasing preference for the first, third, and second N-glycosylation sites was observed in alpha(1)-antitrypsin. This multiple reaction monitoring LC-MS/MS method can be extended to multiple glycoproteins, thereby enabling a glycoproteomics survey of N-glycosylation site occupancies in biological samples.