Early treatment is key to recovery, but this is hampered by the pathology of the disease itself, which mimics the hepatic fibrosis seen with cirrhosis due to HCV progression. Circulating biomarkers currently available, such as alpha-fetoprotein (AFP), are not sensitive or precise enough for definitive identification of HCC in patients.
Mustafa and co-authors (2013) have developed what they term a “discovery and validation strategy” for pulling out novel potential biomarkers to distinguish HCC from HCV.1 Their approach uses various mass spectrometry (MS) proteomics techniques, including liquid chromatography tandem MS (LC-MS/MS), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, selective reaction monitoring (SRM) and differential stable isotope labeling.
The researchers analyzed serum samples from two patient groups with confirmed HCV infection and either positive or negative for histologically verified HCC. Using an aptamer-based serum enrichment technique to overcome potential masking by the complex serum proteome, Mustafa et al. examined the samples for differential protein expression using two-dimensional difference gel electrophoresis (2D-DIGE). They identified several differentially expressed candidates using this method, but the research team chose to focus further validation efforts on the novel candidate apolipoprotein A1 (ApoA1).
Samples were prepared for mass spectrometric analysis using isoelectric focusing in conjunction with 2D-polyacrylamide gel electrophoresis to isolate the relevant protein spots. The region of interest was removed from the gel and digested with trypsin before undergoing MALDI-TOF MS and MALDI-MS/MS. The researchers further confirmed the identification of ApoA1 after they performed sample analysis with nano-LC-MS/MS (LTQ Orbitrap Velos, Thermo Scientific) and evaluated the data using Proteome Discoverer (Thermo Scientific).
After confirming the identity of ApoA1 as the biomarker of interest, Mustafa et al. used differential stable isotope labeling and SRM to quantify protein levels. The digested samples were incubated with 18O/16O and analyzed in an LTQ linear ion trap mass spectrometer (Thermo Scientific) in conjunction with a ProteomX® nano-HPLC system (Thermo Scientific). Using triple quadrupole MS and targeted candidate ApoA1 peptide fragments by SRM, and against isotopically labeled internal AQUA peptide standards (Thermo Scientific), the researchers also calculated the relative quantities of the proteins. These assays confirmed that the reduction in serum ApoA1 levels in HCC patients was reproducible under the different proteomic techniques, enabling accurate identification and quantification of the putative biomarker.
Finally, the research team confirmed differential expression of ApoA1 immunologically by western blotting, where HCC concentrations of the new biomarker were shown to be 69% of HCV values.
Although results indicate that ApoA1 concentrations are lower in serum from HCV patients with HCC, the researchers acknowledge that results were not clear enough to confirm ApoA1 as a sole indicator of the disease. They call for continued MS-based research utilizing a panel of biomarkers that includes ApoA1 for the discovery of other circulating proteins that may be diagnostic for HCC.
Mustafa, G.M. (2013, September 5) “Biomarker Discovery for Early Detection of Hepatocellular Carcinoma (HCC) in Hepatitis C (HCV) Infected Patients,” Molecular Cell Proteomics [e-pub ahead of print].
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