Multiplexing analysis of serum proteins for potential biomarkers in patients with hepatocellular carcinoma

초록

Background: The high mortality associated with Hepatocellular carcinoma (HCC) is partly due to diagnosis of HCC at advanced stage where effective therapies are lacking. Serum AFP, 72kDa glycoprotein, is of limited value in early detection of HCC and often elevated in non-cancerous condition. In recent advance in mass spectrometry based multiplexed targeted protein quantification facilitates the discovery of potential biomarkers from large number of clinical samples in a high-throughput manner and patient’s friendly way. Aims: Aims of our study were to analyze the serum proteins and to identify the HCC specific potential serum biomarkers for the early detection of HCC using multiplexed proteomic techniques such as nano-LC/MS/MS and MRM (multiple reaction monitoring) in HBV infected HCC patients. Methods: Sera from 50 patients with liver cirrhosis and HCC were used for analysis. Differentially expressed proteins in HCC patients were identified through the intensive protein profiling of serum proteomes using nano-LC/MS/MS and the selected multiple biomarkers including AFP, golgi protein 73, glypican 3, osteopontin, HSP 27 were validated quantitatively and simultaneously by using triple quadruple mass spectrometer. Results: A total of 243 proteins were identified and quantified in the sera of HCC patients, of which 41 proteins and 19 proteins showed abundance changes of at least two fold elevations or reduction, respectively in the pooled sera of HCC compare to liver cirrhosis. Through the gene ontology analysis of differentially expressed proteins in HCC patients, many of the identified proteins were categorized into the inflammatory response, wound healing, acute phase response, defense response, response to stress, immune process, cellular developmental process, and complement activation. Selected target proteins among the differentially expressed proteins were validated with 50 cases of patients’ sera by MRM method. Conclusion: With the advantage of multiplexing proteomic approach and MRM, we discovered several serum biomarker candidates associated with HCC, which could be developed as potential biomarkers for early diagnosis of HCC.