Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma
Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma
复制标题
DOI：
10.1007/s00216-019-01775-5
复制
发表时间：
2019-05-01
影响因子：
4.3
通讯作者：
Yoo, Jong Shin
中科院分区：
文献类型：
--
作者：
Kim, Kwang Hoe;Park, Gun Wook;Yoo, Jong Shin
蛋白质的N-糖基化是与多种生物学功能相关的最重要的翻译后修饰之一。由于N-糖蛋白的分析灵敏度和选择性较低，因此在液质联用(LC-MS)中对其进行鉴定和定量是一项具有挑战性的工作。这是由于它们的微观异质性和合成N-糖肽作为内标的困难。平行反应监测(PRM)在靶向LC-MS中得到广泛应用。LC-PRM的主要优点是，即使没有内标，它也可以利用串联质谱仪(MS/MS)裂解来鉴定N-糖肽。我们研究了利用多重免疫沉淀分析N-糖蛋白以提高灵敏度和选择性的可行性。我们针对在肝细胞癌中异常糖化的N-糖蛋白[-甲胎蛋白(AFP)、玻璃体连结蛋白(VTN)和-1-抗糜蛋白酶(AACT)]。选择它们的胰酶N-糖肽，用Y离子测定岩藻糖化N-糖肽的百分比，其中包括带有氨基酸序列的糖肽片段。最后，我们证实AFP和VTN联合使用的受试者工作特征曲线下面积(AUC=0.944)比单一糖肽(AUC值分别为0.889和0.792)在区分肝细胞癌和肝硬变血清方面增加得更多。本研究表明，从血清中免疫沉淀的多重N-糖蛋白的LC-PRM方法可以应用于癌症生物标志物的开发和验证。
The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in liquid chromatography-mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins [-fetoprotein (AFP), vitronectin (VTN), and -1-antichymotrypsin (AACT)] that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC=0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC=0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers.