Direct Monitoring of Fucosylated Glycopeptides of Alpha-Fetoprotein in Hepatocellular Carcinoma Serum by LC-MS/MS with Immunoprecipitation
Kwang Hoe Kim (1), Soo-Youn Lee (2), Gun Wook Park (1), Eun Sun Ji (1), Jin Young Kim (1), * and Jong Shin Yoo (1, 3),* (1) Biomedical Omics Group, Korea Basic Science Institute, Republic of Korea. (2) Department of Laboratory Medicine and Genetics, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. (3) Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Republic of Korea.
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Kwang Hoe Kim (Presenter) Korea Basic Science Institute
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction: The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various cancers. Among them, alpha-fetoprotein (AFP) is a widely used serological marker that has been associated with hepatocellular carcinoma (HCC). Even though the level of AFP is increased in serum of HCC patients, its diagnostic sensitivity of HCC is poor because AFP levels are also increased in liver diseases, such as cirrhosis. Therefore, various assays for HCC have been developed to increase the sensitivity and selectivity for diagnosis. Above all, changes of fucosylation have been reported to be associated with the development of HCC.
Objectives: We directly monitored fucosylated glycopeptides in AFP to distinguish between HCC and cirrhosis patients by parallel reaction monitoring (PRM) mass spectrometry (MS) combined with immunoprecipitation. We sought to evaluate glycopeptides of AFP as a tool for differentiating between early HCC and cirrhosis.
Methods: First, LC-MS/MS-based PRM combined with immunoprecipitation was performed to analyze glycopeptides. Second, sialic acid was removed enzymatically using a neuraminidase to improve the analytical sensitivity. Finally, tryptic N-glycopeptides were selected to determine the percentages of fucosylated AFP using Y ions, which include glycopeptide fragments with amino acid sequences.
Results: The treatment of neuraminidase to glycopeptides for desialylation was useful to improve MS detection limit (LOD < 2 ng/mL) and to obtain reliable signal (CV < 20%) of target glycopeptides in AFP from sub µL serum. Finally, relative percentage of fucosylated AFP (AFP-fuc%) out of total glycosylated one was applied to compare sera with HCC and liver cirrhosis. AFP-fuc% showed an area under the ROC curve (AUC = 0.949) to discriminate between HCC and cirrhosis patients.
Conclusion: This study provides a promising analytical strategy for direct monitoring of relative percentage of fucosylated glycopeptides without internal standard by combining a LC-PRM with immunoprecipitation. Thus, our method may have wide applications for the verification of glycoprotein biomarkers in blood samples.
= Discovery stage.
= Translation stage.
= Clinically available.
