Phospho zinc finger protein: A promising serum biomolecule as noninvasive diagnostic marker of chronic Hepatitis B related liver diseases including liver cancer

Abstract

Context: Liver cancer or hepatocellular carcinoma (HCC) is a dreadful complex disease generally occurring from chronic hepatitis B (HBV-CH) due to its latency, which leads to liver cirrhosis and ultimately liver cancer. To prevent cancer at root level, diagnosis of HBV-CH is highly necessary which based on clinical presentation, serum biochemistry, and viral markers. Aim: The aim of the present study was to detect and identify phosphorylated proteins in HBV-CH patients' sera, among chronic hepatitis B-induced liver cirrhosis (HBV-LC) and HCC by developing antibody against this targeted phosphoprotein by enzyme-linked immunosorbent assay (ELISA). This candidate phosphoprotein in patients' sera can be a noninvasive biomarker of HBV-CH. Setting and Design: Our experimental approach was to detect phosphoproteins in HBV-CH, HBV-LC, and HCC, their quantification by ELISA and Western blot. Identification of highly expressed targeted phosphoproteins was done by two-dimensional (2D) gel electrophoresis followed by MALDI-ToF-MS analysis. Antibody is to be developed against synthesized peptide of targeted phosphoprotein of HBV-CH to use by ELISA. This will be a non-invasive approach to identify candidate phosphoprotein as biomarker of HBV-CH. Methodology: Our experimental approach consisted of three steps: (1) detection of serum phosphoproteins by Pro-Q diamond dye in HBV-CH, HBV-LC and HCC patients' groups as well as control subjects; (2) quantification of serum phosphoproteins using different phospho-specific monoclonal antibodies viz., antiphosphoserine (pSer), antiphosphothreonine (pThr), and antiphosphotyrosine (pTyr) antibodies by ELISA and Western blot; (3)identification of differentially expressed phosphorylated proteins in HBV-CH, HBV-LC and HCC by 2D electrophoresis (2DE) followed by in gel trypsin digestion and subsequently by MALDI-ToF-MS analysis. Statistical Analysis Used: Student's t-test and ANOVA was applied for statistical analysis. Results: There were four phosphoprotein bands namely at 25, 50, 70, 75 kDa in HBV-CH, HBV-LC, HCC and control subjects detected by ProQ diamond dye. Besides there appeared one more band at 60 kDa in HCC. The phosphorylation level at serine and threonine residues was highest in HCC patient groups among HBV-CH, HBV-LC and control groups whereas no phosphorylation level of tyrosine was observed among liver disease patient and control groups. Serum phosphorylated proteins were detected and quantified by Western blot. The results were corroborated to those obtained by ELISA. The differential expression of seven phosphoprotein spots was detected in HBV-CH, HBV-LC, HCC patients and control subjects by 2DE and were identified by MALDI-ToF-MS analysis. Conclusion: Thus circulating phosphoproteins could represent important disease biomarkers because of their differential expression in liver diseases.

Keywords

• biomarkers
• enzyme-linked immunosorbent assay
• liver disease
• phosphoproteins
• western blot