title: New approaches for biomarker discovery: the search for liver fibrosis markers in hepatitis C patients.
abstract: Despite many shortcomings, liver biopsy is regarded as the gold standard for assessing liver fibrosis. A less invasive and equally or more reliable approach would constitute a major advancement in the field. Proteomics can aid discovery of novel serological markers and these proteins can be measured in patient blood. A major challenge of discovering biomarkers in serum is the presence of highly abundant serum proteins, which restricts the levels of total protein loaded onto gels and limits the detection of low abundance features. To overcome this problem, we used two-dimensional gel electrophoresis (2-DE) over a narrow pH 3-5.6 range since this lies outside the range of highly abundant albumin, transferrin and immunoglobulins. In addition, we used in-solution isoelectric focusing followed by SDS-PAGE to find biomarkers in hepatitis C induced liver cirrhosis. Using the pH 3-5.6 range for 2-DE, we achieved improved representation of low abundance features and enhanced separation. We found in-solution isoelectric focusing to be beneficial for analyzing basic, high molecular weight proteins. Using this method, the beta chains of both complement C3 and C4 were found to decrease in serum from hepatitis C patients with cirrhosis, a change not observed previously by 2-DE. We present two proteomics approaches that can aid in the discovery of clinical biomarkers in various diseases and discuss how these approaches have helped to identify 23 novel biomarkers for hepatic fibrosis.
authors.affiliation: Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom. Bevin.Gangadharan@bioch.ox.ac.uk
authors.forename: Bevin
authors.initials: B
authors.lastname: Gangadharan
authors.forename: Robin
authors.initials: R
authors.lastname: Antrobus
authors.forename: David
authors.initials: D
authors.lastname: Chittenden
authors.forename: Jan
authors.initials: J
authors.lastname: Rossa
authors.forename: Manisha
authors.initials: M
authors.lastname: Bapat
authors.forename: Paul
authors.initials: P
authors.lastname: Klenerman
authors.forename: Eleanor
authors.initials: E
authors.lastname: Barnes
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: Nicole
authors.initials: N
authors.lastname: Zitzmann
mesh_terms: D015415:Biomarkers / Q000097:blood*; D003179:Complement C3b / Q000378:metabolism; D015935:Complement C4b / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006526:Hepatitis C / Q000150:complications*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008103:Liver Cirrhosis / Q000097:blood* / Q000175:diagnosis* / Q000209:etiology; D040901:Proteomics / Q000379:methods*
chemical_list: D015415:Biomarkers; D003179:Complement C3b; D015935:Complement C4b