Shotgun proteomics analysis of differentially expressed urinary proteins involved in the hepatocellular carcinoma

Abstract

Numerous investigations underlying the hepatocellular carcinoma (HCC) diagnosis as well as detection at an early stage based on hepatitis B virus surface antigen (HbsAg) concentration in serum and aflatoxin metabolites in urine have been commonly reported in the literature. To date, these biomarkers, even though specific and accurate, are not universal for the detection of HCC elicited by all causative factors. In addition, potential biomarkers may be present at low concentrations in contrast to the presence of abundant interfering proteins with a wide dynamic range. The aim of this study is to establish an operational analysis platform of effective and noninvasive diagnostic tool with a high sensitivity to explore protein expression profiles by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry (nanoLC-MS/MS) and stable isotope dimethyl labeling. Differentially expressed urinary proteins were identified and compared by the mass spectral patterns of their peptide fragments generated from protease digestion. In our results, the quantitative proteomic analysis of the differentially expressed proteins in urine identified at least 21 protein biomarker candidates with high confidence levels. We have further identified 14 proteins with up-regulation (stable isotope D/H ratio ≥ 1.5) and 7 with down-regulation (D/H ratio ≤ 0.6). The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of liver throughout progressive developments of HCC. The results would place a firm foundation for future work regarding validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various classes of HCC. © 2014 Kuo CJ, et al.