Quantification of Intact O-Glycopeptides on Haptoglobin in Sera of Patients With Hepatocellular Carcinoma and Liver Cirrhosis

Hong Shu; Lei Zhang; Yiwei Chen; Yijie Guo; Limin Li; Fanghua Chen; Zhao Cao; Guoquan Yan; Chunlai Lu; Chao Liu; Shu Zhang

doi:10.3389/fchem.2021.705341

Quantification of Intact O-Glycopeptides on Haptoglobin in Sera of Patients With Hepatocellular Carcinoma and Liver Cirrhosis

Front Chem. 2021 Jul 14:9:705341. doi: 10.3389/fchem.2021.705341. eCollection 2021.

Authors

Hong Shu^{1

2}, Lei Zhang³, Yiwei Chen⁴, Yijie Guo⁵, Limin Li⁶, Fanghua Chen¹, Zhao Cao⁶, Guoquan Yan³, Chunlai Lu⁴, Chao Liu⁵, Shu Zhang¹

Affiliations

¹ Liver Cancer Institute, Zhongshan Hospital, and Key Laboratory of Carcinogenesis and Cancer Invasion (Ministry of Education), Fudan University, Shanghai, China.
² Department of Clinical Laboratory, Cancer Hospital of Guangxi Medical University, Nanning, China.
³ Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
⁴ Department of Thoracic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China.
⁵ Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, School of Medicine and Engineering, Beihang University, Beijing, China.
⁶ Department of Clinical Laboratory, First Affiliated Hospital of Guangxi Medical University, Nanning, China.

Abstract

Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC. Furthermore, label-free quantification based on MS was performed. A total of 26 intact O-glycopeptides were identified on Hp, and most of them were elevated in HCC as compared to LC. Among them, the intensity of HYEGS ³¹⁶TVPEK (H1N1S1) on Hp was the highest in HCC patients. Increased HYEGS ³¹⁶TVPEK (H1N1S1) in HCC was quantified and confirmed using the MS method based on ¹⁸O/¹⁶O C-terminal labeling and multiple reaction monitoring. This study provided a comprehensive understanding of the glycosylation of Hp in liver diseases.

Keywords: O-glycosylation; haptoglobin; hepatocellular carcinoma; lectin; mass spectrometry.