University of Texas Medical Branch, United States;
Chronic hepatic disease damages the liver, and the resulting wound healing process lead to liver fibrosis and subsequent development of cirrhosis. The leading causes of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis 2to5% develop Hepatocellular carcinoma (HCC) with survival rate of 7%. HCC is one of the leading causes of cancer related deaths worldwide and the poor survival rate is largely due to late stage diagnosis making successful intervention difficult if not impossible. The lack of sensitive and specific diagnostic tools, and urgent need for early stage diagnosis, prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer based fractionation technology to reduce serum complexity, differentially labeled samples (6 HCC, 6 HCV) with fluorescent dyes, and resolved proteins in pair-wise 2D-DIGE. DeCyder software was used to identify differentially expressed proteins, spots picked, and MALDI/MS/MS used to identify that ApoA1 was down regulated by 22% (p<0.004) in HCC compared to HCV. Differential expression quantified by 2D DIGE was confirmed by 18O/16O stable isotope differential labeling with LC/MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC/MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, VSFLSALEEYTK) using 18O/16O labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays which were independently confirmed by western blotting. Thus, ApoA1 is a candidate member of a SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future, multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.