Hepatitis B Fibrosis Stage Associated Plasma Markers Of Quantitative Proteomics Research

LF (Liver/Hepatic fibrosis) is common to many chronic liver diseases of different etiologies, which could lead to cirrhosis and its related complications. 3 - 10% of cirrhotic patients with chronic hepatitis B (CHB) or chronic hepatitis C (CHC) develop primary liver cancer each year. LF is reversible. The accurate assessment of fibrosis in hepatic diseases provides much information and has a high value for the diagnosis and prognosis of disease, for the therapeutic decision and for the monitoring of the natural history or the evolution under treatment. Histological diagnosis with liver biopsy has long been the gold standard for assessing the degree of fibrosis, but it is an invasive procedure with inherent risk and poorly accepted by patients.An ideal noninvasive diagnostic test for hepatic fibrosis should be accurate, readily available, simple and inexpensive. More devotion should be taken to the field of noninvasive diagnostic test for LF or cirrhosis. Plasma-based tests of LF have attracted more attention in recent years. Human plasma is potentially the single most informative sample that can be collected easily and uninvasively from an individual, which describes their current state of health. Till now, some plasma biomarkers have been reported or applied for LF staging, but none of them is accurate or reliable enough in predicting LF. Using quantitative proteomics technology, we can globally examine HBV infected plasma samples from the LF patients who have been assessed by liver biopsy, and search for LF-associated proteins that can be used as candidate biomarkers for diagnosis and/or target proteins for pathogenetic study.N-Terminal isotope tagging strategy for quantitative proteomics is a suitable way for our research. It is based on a gentle chemical labeling strategy that specifically and differentially labels the N-terminus of all peptides in a sample with either a d₅- or d₀-propionyl group. Lysine side chains are blocked by guanidination prior to N-terminal labeling to prevent the incorporation of multiple labels. MS-based quantitation of protein abundance via stable isotope labeling of peptides from enzymatic digests has been carried out on LC- FTICR instruments.As a final result, there are 1,063 plasma proteins identified with quantitative information and totally 246 differently altered protein (Ratio>1.5, P<0.05) among the 5 different Stage (S) groups (SO, S1, S2, S3, S4). Cluster analysis results show 5 different kinds of tendency, inlcuding gradually up-regulated, down-regulated, firstly up and then down-regulated, or firstly down and then up-regulated, and up-down-regulated frequently. Also, the severe fibrosis groups (S3, S4) were clustered in one group compared to the other slight fibrosis groups (S0,S1,S2), show more parallel with the liver fibrosis development. Ingenuity pathway analysis for all differented proteins show that there are 209 protein located into 8 kinds of pathways(Hematological System Development/Lipid metabolism/Cell division &development/Immune & Lymphatic System/Cell Signal& tissue development/Amino acid metabolism /Transcriptional control /Cell division) constructed according to the literature, and totally a whole inter-action map was constructed for all different proteins. It means the proteins have more relation or interaction during the liver fibrosis development. Western blot verification indicated that the quantitative information is not only true, but also show potential and valuable of the candidate biomarkers, such as al-acid-glycoprotein, CDC23, fibronictin etc. for the further investigation and clinical application.²...