| Primary liver cancer is the third most common cause of death from cancer.It has ranked the second highest cancer killer in China and more than half of the new cases occur in our country every year throughout the world.The prognosis of hepatocellular carcinoma(HCC) remains very poor.Hepatectomy and liver transplantation(LTx) have been considered as the main curative treatment.For the patients with liver cancer,LTx allows removing the entire tumor and the cirrhotic liver,and avoids the recurrence.Therefore,LTx is considered as the best therapeutic option for cirrhosis-associated HCC.However,tumor recurrence and metastasis after LTx still remain a significant problem in patients with HCC.Although in the past few years,there has been ongoing development in tumor related characteristics,such as the number and size of nodules,micro/macroscopic vascular invasion,and high serum alpha feto-protein(AFP) levels,which are still insufficient for recognizing patients at high risk for recurrence and selecting those at low risk.In order to improve the diagnosis and prognosis of HCC,it is mandatory to identify molecular tumor markers predictive of recurrence that can define a subset of HCC patients that stand to benefit from LTx.The recent developments of proteomic technology allow us understanding the mechanisms of hepatocarcinogenesis and discovering diagnostic markers and therapeutic targets for HCC.Development of new technology for identification of tumor-related molecular markers is currently in progress.However,proteomics studies are still needed to explore the recurrence and metastasis of HCC after LTx.As the most available sample,serum is widely applied for early diagnosis and treatment of diseases in clinical research.Serum offers particularly promising resources for biomarker discovery.Meanwhile the complex nature of serum,with the enormous dynamic range of concentration at 10 orders of magnitude,makes detection of low-abundance disease biomarkers very challenging.In our study,the effective and repeatable system,MARS HPLC column (Agilent Technologies,Wilmington,DE,USA)(4.6mm×100mm),was applies to remove the high-abundance proteins.The HPLC column contains polyclonal antibodies to human albumin,transferring,IgG,IgA,haptoglobin and antitrypsin etc. The HPLC column removed about 94%of the total protein in human serum and this reduced complexity enabled the low-abundant proteins bands unmasked by the high-abundant proteins.The lanes in SDS-PAGE map were clearer and some low-abundance proteins were visualized after depletion.The effective method of removing high-abundance proteins in serum is established,which providing basis for further strategies in proteomics research.Two-dimensional electrophoresis(2-DE) is a prominent separation method in traditional proteomics.Total protein was quantified using the dye-binding Coomassie protein assay,which may interfere with the following MS.Moreover,standard 2-DE gels do not reflect a true representation of hydrophobic,highly insoluble,very basic, as well as very small and very large proteins.Therefore 2-DE is unsuitable for further analysis in the research of proteomics.Differential tagging with various isotopic reagents,such as iTRAQ(isobaric tag for relative and absolute quantitation), followed by multidimensional LC and MS/MS analysis is emerging as one of the more powerful methodologies in the search for disease biomarkers.Through the use of amine-specific isobaric tags,the iTRAQ technology facilitates the simultaneous analysis of up to four different conditions of interest in the same iTRAQ experiment (ABI developed 8-plex iTRAQTM kits in 2007).This technology simplifies the workflow allowing rapid progression to LC/MS/MS analysis and easy data interpretation,expanding proteome coverage by labeling all peptides,including those with post-translational modifications(PTMs) to extract more detailed information from samples.In our research,sera from HCC patients undergoing LTx in our hospital with complete clinicopathological and follow-up data,within UCSF criteria and sharing a similar disease background were categorized into two groups:recurrence and non-recurrence groups.Samples were immunodepleted for 14 highly abundant serum proteins,and then labled with mass-balanced isobaric tags(iTRAQ),and analyzed by liquid chromatography-tandem mass spectrometry(LC-MS/MS). Totally 95 proteins with quantitation information were identified.Forty-eight of these proteins(50.5%) were displayed differentially in the recurrent group, compared to the DFS group.Among these 48 proteins,13 were found to be up-regulated(>1.2-folds),11 were down-regulated(<0.8-folds).All these proteins were classified according to their molecular function,using the tools at www.geneontology.org.Which were classified into eight groups:binding activity (32.56%),enzyme activity(25.58%),lipid metabolism(9.30%),transporter activity (6.98%),immune function(6.98%),complement function(2.33%),antioxidant activity(2.33%),and others(13.95%).The results implied that iTRAQ combined quantitative proteomics have provided a special protein expression patterns in metastatic and recurrent states of HCC patients undergoing LTx.These differential proteins maybe serve as new prognostic biomarker and potential therapeutic targets particularly for HCC patients undergoing LTx,which warrants further investigation.POTENTIAL APPLICATIONiTRAQ combined quantitative proteomics have provided a special protein expression patterns in metastatic and recurrent states of HCC patients undergoing LTx.These differential proteins maybe serve as new prognostic biomarkers and potential therapeutic targets particularly for HCC patients undergoing LTx.NOVELTYWe obtained quantitative protein expression profiles of primary tumor by 2D-LC-MS/MS combined with iTRAQ,which may overcome the difficulties by traditional techniques.Furthermore,the approach to screen the key differential proteins sets the base for the novel and promising prognostic biomarkers and therapeutic targets after LTx and thereby improves the therapeutic effect of HCC. |
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