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Anal Bioanal Chem. 2019 May;411(14):3009-3019. doi: 10.1007/s00216-019-01775-5. Epub 2019 May 11.

Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma.

Author information

1
Biomedical Omics Research Group, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongju, 28119, Republic of Korea.
2
Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 34134, Republic of Korea.
3
Biomedical Omics Research Group, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongju, 28119, Republic of Korea. jinyoung@kbsi.re.kr.
4
Biomedical Omics Research Group, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongju, 28119, Republic of Korea. jongshin@kbsi.re.kr.
5
Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 34134, Republic of Korea. jongshin@kbsi.re.kr.

Abstract

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 [α-fetoprotein (AFP), vitronectin (VTN), and α-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. Graphical abstract.

KEYWORDS:

Hepatocellular carcinoma; Immunoprecipitation; Internal standard; N-Glycopeptide; Parallel reaction monitoring

PMID:
31076819
DOI:
10.1007/s00216-019-01775-5

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