Comparative Proteome Research In Serum AFP Negative Hepatocellular Carcinoma Cases

Introduction:Hepatocellular carcinoma(HCC) ranks fifth in cancer frequency in the world with an estimated 1 million new cases occurring per year.Early detection is one of the key strategies in reducing liver cancer death.40%of these HCC cases show serum AFP negative.It is urgent need great to acknowledge the molecular mechanism and identify additional novel HCC markers for early detection of liver cancer in the AFP-negative cases.Experimental procedure:In this study,focus on the AFP negative cases,we utilize 1 D SDS-PAGE coupling with LC ESI-MS/MS label free shotgun strategy to analyze the tissue proteome of the cancerous and adjacent normal tissues,the tissue secretome of the cancerous and adjacent normal tissues,the plasma proteome of the AFP negative HCC cases and health controls.With the full proteomics research,we can acknowledge the molecular mechanism and reveal the potential biomarker candidates in the AFP negative HCC cases.To the tissue proteome,we utilize 1 D SDS-PAGE coupling with nanoUPLC ESI MS/MS((Q-TOF primier),perform the proteome research of 9 paired cancerous and their adjacent normal tissues.The search engine in this study is PLGS 2.3.To the tissue secretome,we culture the tissues in serum free media,then utilize 1 D SDS-PAGE coupling with MDLC ESI MS/MS(LTQ-Orbitrap),perform the secretome research of 4 paired cancerous and their adjacent normal tissues.The search engine in this study is SEQUEST.To the plasma proteome,we perform abundant protein fraction using immnoaffinity method,then utilize 1 D SDS-PAGE coupling with nanoUPLC ESI MS/MS((Q-TOF primier ),perform the plasma proteomics research of 3 paired plasma of the AFP negative HCC cases and health controls.The search engine in this study is X! TANDEM.Result:In the tissue proteome,we totally identified 3908 proteins,compared to the paired adjacent normal tissue,in which 282 proteins over-expressed at least 2 folds,and 127 proteins down-expressed at least 2 folds.The differences were validated by western blot.In the tissue secretome,we totally identified 1302 proteins,compared to the paired adjacent normal tissue,in which 176 proteins over-expressed at least 2 folds,and 200 proteins down-expressed at least 2 folds.The differences were validated by western blot.In the plasma proteome,we totally identified 2779 proteins,compared to the paired health controls,in which 107 proteins over-expressed at least 2 folds,and 102 proteins down-expressed at least 2 folds.Conclusion:For us,this is the first time to research the tissue proteome,tissue secretome and plasma proteome simultaneously,we have found a complete proteome database for AFP negative HCC cases,which enable us perform the further functional and applicable research.