Targeted Mass Spectrometric Approach for Biomarker Discovery and Validation with Nonglycosylated Tryptic Peptides from N-linked Glycoproteins in Human Plasma
Targeted Mass Spectrometric Approach for Biomarker Discovery and Validation with Nonglycosylated Tryptic Peptides from N-linked Glycoproteins in Human Plasma
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DOI：
10.1074/mcp.m111.009290-1
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发表时间：
2011-12-01
影响因子：
7
通讯作者：
Yoo, Jong Shin
中科院分区：
文献类型：
--
作者：
Lee, Ju Yeon;Kim, Jin Young;Yoo, Jong Shin
一种用于发现和验证人类血浆中生物标志物的简单质谱方法是针对n -连接糖蛋白中糖基化位点附近的非糖基化胰蛋白酶肽，这是早期检测、预后和疾病治疗中最重要的生物标志物之一。新型生物标志物的发现和验证需要复杂的样品预处理步骤，例如耗尽高丰度蛋白质，富集所需蛋白质或开发新抗体。目前的研究利用了n -连接糖蛋白中聚糖单元的空间位阻，如果酶活性氨基酸靠近n -连接的糖基化位点，这将显著影响蛋白水解消化的效率。根据糖基化程度的不同，蛋白水解消化会产生数量不同的肽产物。首先用α -1-酸糖蛋白（AGP）作为模型化合物与去糖基化α -1-酸糖蛋白对比，证明了多糖位阻对胰蛋白酶消化的影响。其次，非糖基化胰蛋白酶生物标记物在质谱分析中通常比糖基化胰蛋白酶生物标记物具有更高的灵敏度，使用无标记方法在人肝癌血浆中进行定量，不需要n -连接糖蛋白富集。最后，通过多反应监测分析验证了该方法，表明新发现的非糖基化胰蛋白酶靶点在正常和肝癌血浆中以不同水平存在。由玻璃体粘连蛋白前体蛋白分析非糖基化色氨酸生成的受者工作特征曲线下面积为0.978，在肝癌患者组中观察到最高。这项工作提供了一种有针对性的方法来发现和验证非糖基化胰蛋白酶肽作为人血浆中的生物标志物，而不需要复杂的富集过程或昂贵的抗体制备。中国生物医学工程学报（自然科学版），2011,31(2):444 - 444。
A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations. Molecular & Cellular Proteomics 10: 10.1074/mcp.M111.009290, 1-12, 2011.