Bevin Gangadharan
University of Oxford, UK
Title: Discovery and quantitation of novel fatty liver and fibrosis biomarkers using proteomics
Biography
Biography: Bevin Gangadharan
Abstract
Background
Proteomics can help to discover and quantify novel biomarkers for pancreatic cancer and liver diseases. We show how this is achieved using non-alcoholic fatty liver disease (NAFLD) as an example. For diagnosis and staging of NAFLD, liver biopsy is the reference standard. Less invasively serum biomarkers can be detected using immunoassays. However biomarkers can degrade due to sample storage conditions and therefore may not be detected using these antibody-based assays. This disadvantage can be overcome by using mass spectrometry which is antibody-free.
Methods
Novel biomarkers were identified by analysing proteins in serum samples from healthy individuals and patients with varying stages of NAFLD using two proteomics techniques: mass spectrometry (MS) and two dimensional gel electrophoresis (2DE). For the most promising biomarkers, an antibody-free assay (parallel reaction monitoring using mass spectrometry) was used which detects tryptic peptides of the biomarkers and their fragments. A calibration curve, established from known amounts of synthetic isotopically labelled peptides, was used to determine the concentrations of our biomarkers in serum/plasma samples from patients with varying stages of NAFLD.
Results
Several candidate biomarkers for NAFLD were identified using MS and 2DE. Our best biomarkers were promising when compared to the proteins used in existing serum biomarker tests. Using parallel reaction monitoring, antibody-free assays were developed for our most promising biomarkers which were able to successfully discriminate between neighbouring NAFLD stages.
Conclusion
We have developed a fast, sensitive and robust antibody-free method to detect and quantify novel NAFLD biomarkers in human serum. Unlike immunoassays which are restricted on the number of biomarkers due to antibody cost, our method can successfully detect and quantify more than 50 biomarkers in a single run. Our assay is nine times faster than conventional quantitation by MS making our approach for absolute biomarker quantitation applicable for clinical use. This is also the only assay which can analyse all points of the calibration curve and determine the absolute concentration of the biomarker in a single acquisition. This novel assay may help clinicians to assess NAFLD and reduce the need for invasive liver biopsies.