Identification of N-Glycosylation Sites on Secreted Proteins of Human Hepatocellular Carcinoma Cells with a Complementary Proteomics Approach
Identification of N-Glycosylation Sites on Secreted Proteins of Human Hepatocellular Carcinoma Cells with a Complementary Proteomics Approach
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利用补充蛋白质组学方法鉴定人肝细胞癌细胞分泌蛋白上的 N-糖基化位点
DOI：
10.1021/pr800826u
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发表时间：
2009-02-01
影响因子：
4.4
通讯作者：
Yang, Pengyuan
中科院分区：
文献类型：
--
作者：
Cao, Jing;Shen, Chengping;Yang, Pengyuan
N-连接糖基化在进入细胞外环境的蛋白质中普遍存在；几乎所有分泌的蛋白质都是糖基化的。然而，关于它们的糖基化位点却很少受到关注。在这里，我们报告了人肝细胞癌细胞分泌蛋白的 N-糖基化位点的分析。为了富集糖肽，互补使用亲水亲和力 (HA) 和酰肼化学 (HC) 捕获方法。通过将这两种方法与 Nano-LC-ESI-MS/MS 分析相结合，鉴定出 194 个独特糖蛋白内的 300 个不同糖基化位点，其中 172 个糖基化位点之前尚未通过实验确定。还首次研究了 HA 和 HC 方法之间的直接比较。简而言之，就糖肽的选择性而言，HC优于HA（92.9% vs 51.3%）；然而，根据已识别的糖基化位点数量，HA 超过 HC（265 比 159）。此外，通过使用这种糖蛋白组学策略，可以轻松消除不可避免的污染物，例如未 N-糖基化的肌动蛋白和牛血清白蛋白。结果，鉴定出了更多低丰度和真正分泌的蛋白质。据报道，在已鉴定的糖蛋白中，甲胎蛋白、CD44 和层粘连蛋白与 HCC 及其转移有关。
N-linked glycosylation is prevalent in proteins destined for extracellular environments; nearly all secreted proteins are glycosylated. However, with respect to their glycosylation sites, little attention has been paid. Here, we report the analysis of N-glycosylation sites on secreted proteins of human hepatocellular carcinoma cells. For the enrichment of glycopeptides, capture methods with hydrophilic affinity (HA) and hydrazide chemistry (HC) were used complementarily. With the use of both methods in combination with nano-LC-ESI-MS/MS analysis, 300 different glycosylation sites within 194 unique glycoproteins were identified, and 172 glycosites have not been determined experimentally previously. A direct comparison between HA and HC methods was also investigated for the first time. In brief, in terms of selectivity for glycopeptides, HC is superior to HA (92.9% vs 51.3%); however, based on the number of glycosites identified, HA outweighs HC (265 vs 159). Furthermore, unavoidable contaminants such as actin and bovine serum albumin which are not N-glycosylated could be easily depleted by using this glycoproteomic strategy. As a consequence, more low-abundance and genuinely secreted proteins were identified. Among the glycoproteins identified, alpha-fetoprotein, CD44 and laminin have been reported to be implicated in HCC and its metastasis.