Tandem Mass Tag-Based Proteomic Analysis of Potential Biomarkers for Hepatocellular Carcinoma Differentiation.PURPOSE
 The poor prognosis of hepatocellular carcinoma (HCC) urgent us to discover early and effective biomarkers.  In this study, we applied tandem mass tag (TMT)-based proteomic analysis to discover potential protein markers for HCC identification and differentiation.


 Fifteen patients, well-differentiated (G1, N = 5), moderate-differentiated (G2, N = 5), and poorly differentiated (G3, N = 5), with 30 matched pair tissues (both tumor and adjacent non-tumor tissues derived from the same patient) were enrolled.  All samples were subjected to TMT labeling and LC-MS/MS analysis.  The identified proteins were subsequently assigned to GO and KEGG for predicting function.  The identified protein candidates were validated using immunohistochemistry (IHC).


 Of these, 154 differentially expressed proteins (DEPs), 100 up-regulated and 54 down-regulated, were found between tumor and adjacent non-tumor tissues; 12 DEPs, 9 up-regulated and 3 down-regulated, were found between G1 and G3 tissues; 8 DEPs, 5 up-regulated and 3 down-regulated, were found between G1 and G2 tissues; 11 DEPs, 8 up-regulated and 3 down-regulated, were found between G2 and G3 tissues.  Among them, ASS1 and CPS1 were significantly up-regulated while UROD and HBB were significantly down-regulated in G3 compared with G1 and G2 tumors.  Three proteins, CYB5A, FKBP11 and YBX1, were significantly up-regulated in G1 compared with both G2 and G3 tumors.  The 7 biomarker candidates were further verified by IHC.


 CONCLUSION
 A variety of DEPs related to the histological differentiation of HCC were identified, among which ASS1, CPS1, URPD and HBB proteins were potential biomarkers for distinguishing poorly differentiated HCC, while CYB5A, FKBP11 and YBX1 were potential biomarkers for distinguishing well-differentiated HCC.  Our findings may further provide a new insight facilitating the diagnosis and prognosis of HCC.
C
All samples were subjected to TMT labeling and LC-MS/MS analysis. 
LC-MS/MS
The identified protein candidates were validated using immunohistochemistry (IHC).


 The 7 biomarker candidates were further verified by IHC.



IHC