Development of a Minimally Invasive Serum Test for Liver Cirrhosis
DOI:
https://doi.org/10.14738/bjhr.1304.11960Keywords:
Phosphorylation, Phospho-Zinc-finger protein, Liver cirrhosis, Biomarker, ELISAAbstract
Phosphorylation is one of the most important post-translational modifications of proteins. Although cellular phosphorylation has been studied extensively, research on phosphoproteomes in patients’ serum with hepatitis is limited. Serum phosphoproteins and phosphopeptides could be considered important disease biomarkers, as they play roles in cellular function. We previously reported higher serum levels of the phosphoZinc finger (pZNF) protein in patients with liver disease, including chronic hepatitis B (CHB), CHB-induced liver cirrhosis (CHB-LC), and CHB-induced hepatocellular carcinoma (CHB-HCC). To develop a minimally invasive pZNF protein-based diagnostic test for liver diseases (CHB, CHB-LC, HCC) from serum, we first synthesized a pZNF peptide based on our MALDI-ToF-MS peptide analysis data and developed polyclonal antibodies specific to pZNF. Using the pZNF-specific antibodies, an optimized Enzyme-Linked Immunosorbent Assay (ELISA) was then developed to screen sera from CHB (n=20), CHB-LC (n=17), CHB-HCC (n=20), and healthy volunteers (n=20). Results from the optimized ELISA test showed that sera from patients with liver diseases could be reliably detected, whereas those from healthy individuals could not (87.7% sensitivity and 76.9% specificity at a 300 cut-off; 82.6% sensitivity and 90.9% specificity at a 400 cut-off). Interestingly, the CHB-LC cohort stood out from the rest cohorts as it showed the highest expression of pZNF protein. At an 800-cut-off value, 17 of 17 CHB-LC samples were detected (above the cut-off), yielding a sensitivity of 100%. At the same cut-off value (800), only 2 of 60 samples were false positives, yielding a specificity of 96.8%. The two positive samples were from the CHB cohort, none from the HV cohort. Overall, we successfully developed a serum test (minimally invasive) that could detect CHB and CHB-induced liver diseases, particularly CHB-LC, with 100% sensitivity and nearly 100% specificity.
References
Ghosh, R.; Ahmed, R.; Ahmed, H.; Chatterjee, B.P. (2022). Phosphorylated Proteins from Serum: A Promising Potential Diagnostic Biomarker of Cancer. International Journal of Molecular Science, 23:123-59.
Ardito, F., Giuliani, M., Perrone, D., Troiano, G., & Lo Muzio, L. (2017). The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review). International Journal of Molecular Medicine, 40:271-280.
Anderson NL. (2010). The clinical plasma proteome: A survey of clinical assays for proteins in plasma and serum. Clinical Chemistry, 56:177-85.
Veenstra TD, Conrads TP, Hood BL, Avellino AM, Ellenbogen RG, Morrison RS. Biomarkers: Mining the biofluid proteome. (2005). Molecular and Cellular Proteomics, 4:409-18.
Zhang Q, Faca V, Hanash S. (2011). Mining the plasma proteome for disease applications across seven logs of protein abundance. Journal of Proteome Research, 10:46-50.
Anderson NL, Anderson NG. (2002). The human plasma proteome: History, character, and diagnostic prospects. Molecular and Cellular Proteomics, 1:845-67.
Carrascal M, Gay M, Ovelleiro D, Casas V, Gelpí E, Abian J. (2010). Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines. Journal of Proteome Research, 9:876-84.
Zhou W, Ross MM, Tessitore A, Ornstein D, Vanmeter A, Liotta LA, et al. (2009). An initial characterization of the serum phosphoproteome. Journal of Proteome Research, 8:5523-31.
Nagler RM. (2009). Saliva as a tool for oral cancer diagnosis and prognosis. Oral Oncology, 45:1006-10.
Hu S, Arellano M, Boontheung P, Wang J, Zhou H, Jiang J, et al. (2008). Salivary proteomics for oral cancer biomarker discovery. Clinical Cancer Research, 14:6246-52. Scholar Publishing
de Jong EP, Xie H, Onsongo G, Stone MD, Chen XB, Kooren JA, et al. (2010). Quantitative proteomics reveals myosin and actin as promising saliva biomarkers for distinguishing pre malignant and malignant oral lesions. PLoS One, 5: e11148.
Bahl, J. M., Jensen, S. S., Larsen, M. R., & Heegaard, N. H. (2008). Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry. Analytical chemistry, 80(16):6308-6316.
Hu, L., Zhou, H., Li, Y., Sun, S., Guo, L., Ye, M., Tian, X., Gu, J., Yang, S., & Zou, H. (2009). Profiling of endogenous serum phosphorylated peptides by titanium (IV) immobilized mesoporous silica particles enrichment and MALDI-TOFMS detection. Analytical chemistry, 81(1):94-104.
Oda, Y., Huang, K., Cross, F. R., Cowburn, D., & Chait, B. T. (1999). Accurate quantitation of protein expression and site-specific phosphorylation. Proceedings of the National Academy of Sciences of the United States of America, 96(12):6591-6596.
Martin K, Steinberg TH, Cooley LA, Gee KR, Beechem JM, Patton WF. (2003). Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics, 3:1128-44.
Schmidt SR, Schweikart F, Andersson ME. (2007). Current methods for phosphoprotein isolation and enrichment. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 849:154-62.
Abramczyk, H., Imiela, A., Brożek-Płuska, B., Kopeć, M., Surmacki, J., & Śliwińska, A. (2019). Aberrant Protein Phosphorylation in Cancer by Using Raman Biomarkers. Cancers, 11(12), 2017
Bhatia, Y., Mondal, G., Islam, S., Ghosh, R., Dutta, S., Ghosh, S., Duseja, A., Panda, C., & Chatterjee, B. P. (2021). Phospho zinc finger protein: A promising serum biomolecule as noninvasive diagnostic marker of chronic Hepatitis B related liver diseases including liver cancer. Journal of Radiation and Cancer Research, 12(4), 147.
Guoqiang, L., Xinran M., Lingyan X.; (2020). The roles of zinc finger proteins in non-alcoholic fatty liver disease. Liver Research, 4(1):35-3.
Guo, Y., Miao, X., Sun, X., Li, L., Zhou, A., Zhu, X., Xu, Y., Wang, Q., Li, Z., & Fan, Z. (2023). Zinc finger transcription factor Egf1 promotes non-alcoholic fatty liver disease. JHEP reports: innovation in hepatology, 5(6), 100724
Seok S. H. (2021). Structural Insights into Protein Regulation by Phosphorylation and Substrate Recognition of Protein Kinases/Phosphatases. Life (Basel, Switzerland), 11(9), 957.
Verhelst, X., Dias, A. M., Colombel, J., Vermeire, S., Vlierberghe, H. V., Callewaert, N., Pinho, S. S. (2020). Protein Glycosylation as a Diagnostic and Prognostic Marker of Chronic Inflammatory Gastrointestinal and Liver Diseases. Gastroenterology. 158(1):95-110
Bose, P, P., Chatterjee, U., Chatterjee, B, P. (2018). Assessment of altered fucosylation of serum α-1-acid glycoprotein in hepatocellular carcinoma patients by gold-nanoparticle aggregation immunoassay. Material Science Research India 15 (3):263-271.
Mandal, G., Saroha, A., Bose, P, P., Chatterjee, B, P. (2016). Altered glycosylation, expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C, hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients. Glycoconjugate Journal, 33:209-218.
Mandal, G.,Yagi, H., Kato, K., Chatterjee, B, P. (2015). Structural heterogeneity of glycoform of α-1 acid glycoprotein in alcoholic cirrhosis patients. Advances in Experimental Medicine and Biology, 842:389-401 Scholar Publishing
Mandal, G., Chatterjee, U., Chatterjee, B, P. (2014). Glycosylation of acute phase proteins: A promising disease biomarker. Proceeding of National Academy of Sciences, India, (Biological Sciences) 84:865-874.
Chatterjee, B, P., Mandal, G., Chatterjee, U., (2011) Reactivity of alpha fetoprotein with lectins in the differential diagnosis of non-malignant and malignant liver disease. Trends in Carbohydrate Research3, No-3:26-31.
A Saroha, A., Biswas, S., Chatterjee, B, P. Das, H. (2011). Altered glycosylation and expression of plasma alpha-1-acid glycoprotein and haptoglobin in rheumatoid arthritis. Journal of Chromatography B, 879:1839-1843.
Mondal, G., Chatterjee, U., Chawla, Y., Chatterjee, B, P. (2011). An evaluation of the differences in the glycosylation of alpha-fetoprotein between hepatitis and liver cirrhosis by lectin-ELISA. Trends in Carbohydrate Research3, No-1:13-20.
Mondal, G., Chatterjee, U., Chawla, Y., Chatterjee, B, P. (2011). Alteration of glycan branching and differential expression of sialic acid on alpha fetoprotein among hepatitis patients. Glycoconjugate Journal, 28:1-9.
Chatterjee, B, P., Chatterjee, U., Mandal, G., (2010). Changes of serum protein glycosylation as marker in the differential diagnosis of hepatitis and liver cirrhosis. Proceedings of National Academy of Sciences, India (Biological Sciences), 80:190-200.
Mondal, G., Chatterjee, U., Das, H, R., Chatterjee, B, P. (2009) Enhancement of alpha -1-acid glycoprotein and fucosylation changes in hepatitis B patients as biomarker in the pathogenesis of the disease. Glycoconjugate Journal, 26:1225-1234.
Ramachandran, P., Xu, G., Huang, H. H., Rice, R,. Zhou, B., Lindpaintner, K., Serie, D., (2022). Serum Glycoprotein Markers in Non-alcoholic Steatohepatitis and Hepatocellular Carcinoma. Journal of Proteome Research, 21 (4):1083-1094
Tissandié, E., Morelle, W., Berthelot, L., Vrtovsnik, F., Daugas, E., Walker, F., Lebrec, D., Trawalé, J. M., Francoz, C., Durand, F., Moura, I. C., Paradis, V., Moreau, R., & Monteiro, R. C. (2011). Both IgA nephropathy and alcoholic cirrhosis feature abnormally glycosylated IgA1 and soluble CD89-IgA and IgG-IgA complexes: common mechanisms for distinct diseases. Kidney international, 80(12):1352-1363.
Osna, N. A., Carter, W. G., Ganesan, M., Kirpich, I. A., McClain, C. J., Petersen, D. R., Shearn, C. T., Tomasi, M. L., & Kharbanda, K. K. (2016). Aberrant post-translational protein modifications in the pathogenesis of alcohol-induced liver injury. World journal of Gastroenterology, 22(27):6192-6200.
Cheng, J-Y., Shan, G-Y., Wan, H., Liu, Y-Y., Zhang, Y-X., Shi, W-N., & Li, H-J. (2024) Hepatitis B virus-induced cirrhosis: Mechanisms, global variations, and treatment advances. World J. Hepatol., 16(12): 1515-1523.
Yao, J., Guo, J., & Xie, Y. (2025) Hepatitis B virus induced cirrhosis and hepatocarcinoma: pathogenesis and therapeutics. Explor Dig Dis. 4:100565
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Copyright (c) 2026 Somrita Padma, Rishila Ghosh, Manabendu Barman, Yeshika Bhatia, Ajay Duseja, Sankhadeep Dutta, Chinmoy Kumar Panda, Hafiz Ahmed, Bishnu Pada Chatterjee
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