Development of Glycoprotein Capture-Based Label-Free Method for the High-throughput Screening of Differential Glycoproteins in Hepatocellular Carcinoma
Development of Glycoprotein Capture-Based Label-Free Method for the High-throughput Screening of Differential Glycoproteins in Hepatocellular Carcinoma
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开发基于糖蛋白捕获的无标记方法，用于高通量筛选肝细胞癌中的差异糖蛋白
DOI：
10.1074/mcp.m110.006445
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发表时间：
2011-07-01
影响因子：
7
通讯作者：
Zou, Hanfa
中科院分区：
文献类型：
--
作者：
Chen, Rui;Tan, Yexiong;Zou, Hanfa
开发了一种稳健、可重复且高通量的方法，用于人血清中糖蛋白丰度的相对定量分析。糖蛋白不是通过传统定量糖蛋白组学中的糖肽来定量，而是通过糖蛋白消化物中衍生的非糖基化肽来定量，其中包括捕获血清样品中的糖蛋白，并通过胰蛋白酶消化捕获的糖蛋白，然后通过二维液体释放非糖肽。 释放肽的色谱-串联质谱分析。通过比较不同样品之间已鉴定的糖蛋白的非糖基化肽的光谱计数来实现蛋白质定量。该方法被证明在糖蛋白定量方面具有与糖肽水平捕获几乎相同的特异性和灵敏度。低至每毫升纳克水平的蛋白质差异丰度得到了高置信度的量化。将建立的方法应用于健康人和肝细胞癌（HCC）患者的人血清样本分析，以筛选HCC中的差异糖蛋白。发现正常和 HCC 血清样本之间有 38 种糖蛋白存在显着浓度变化，其中包括甲胎蛋白，这是临床上唯一用于 HCC 诊断的标记物。通过酶联免疫吸附测定进一步证实了与 HCC 发生相关的三种糖蛋白，即半乳糖凝集素 3 结合蛋白、胰岛素样生长因子结合蛋白 3 和血小板反应蛋白 1 的丰度变化。总之，所开发的方法是定量分析人血清中糖蛋白的有效方法，可进一步应用于 HCC 和其他癌症的生物标志物发现。分子与细胞蛋白质组学 10：10.1074/mcp.M110.006445，1-13，2011。
A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including alpha-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.006445, 1-13, 2011.