Division of Mass Spectrometry, Korea Basic Science Institute, Ochang-Myun, Cheongwon-Gun, Republic of Korea.
Mol Cell Proteomics. 2011 Dec;10(12):M111.009290. doi: 10.1074/mcp.M111.009290. Epub 2011 Sep 22.
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.
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，开发了一种简单的用于在人血浆中发现和验证生物标志物的质谱方法，该糖蛋白是非糖基化胰蛋白酶肽是早期检测、预后和疾病治疗的最重要生物标志物之一。发现和验证新的生物标志物需要复杂的样品预处理步骤，如高度丰富蛋白质的耗竭、所需蛋白质的富集或新抗体的开发。本研究利用 N-连接糖蛋白中糖基单元的空间位阻，如果酶活性氨基酸紧邻 N-连接糖基化位点，会显著影响蛋白水解消化的效率。然后，根据糖基化程度，蛋白酶解会产生定量不同的肽产物。首先使用α-1-酸性糖蛋白（AGP）作为模型化合物与去糖基化的α-1-酸性糖蛋白，证明了糖基空间位阻对胰蛋白酶消化的影响。其次，使用无标记方法，无需 N-连接糖蛋白富集，在人肝细胞癌血浆中定量检测非糖基化胰蛋白酶肽生物标志物，该生物标志物在质谱分析中通常比其糖基化对应物具有更高的灵敏度。最后，使用多重反应监测分析对该方法进行了验证，证明了在正常和肝细胞癌血浆中不同水平存在新发现的非糖基化胰蛋白酶肽靶标。通过对来自前 vitronectin 蛋白的非糖基化胰蛋白酶肽的分析生成的接收器操作特性曲线下面积为 0.978，这是在一组肝细胞癌患者中观察到的最高值。这项工作提供了一种靶向方法，用于在人血浆中发现和验证非糖基化胰蛋白酶肽作为生物标志物，而无需复杂的富集过程或昂贵的抗体制备。
