Objective:To investigate proteomics of hepatocellular carcinoma(HCC)cells with different metastatic potentials by isobaric tags for relative and absolute quantitation(i TRAQ);to screen and identify proteins related to metastasis

and to provide new molecular targets for metastasis and treatment. Methods:Cytoplasmic proteins

extracted from three cell lines with similar genetic background and different metastatic potentials were concentrated

digested

desalted and labeled with i TRAQ. Differentially expressed proteins were identified by 2D-LC-MS /MS

then the proteins were annotated by comparing with known protein database. Target proteins related with metastasis were singled out by gene ontology analysis and related pathway analysis using Web Gestalt databases. These proteins were then validated by Western blot. Results:Using i TRAQ labeling for quantification and detection by LC-MS/MS

1 170 proteins were identified. Forty-seven proteins were differential expressed in L/H(MHCC97-L/MHCC9-H)groups

and 65 proteins in M/H(HCCLM3/MHCC97-H)groups. Bioinformatics analysis revealed that differential expression proteins in L/H and M/H groups were mainly related to the catalytic activity of some enzymes and cells adhesion

and play important role in the cellular metabolic process. At translational level

PLEC was up-regulated in MHCC97-H and HCCLM-3

SHMT2 was up-regulated in HCCLM3

LDHA was up-regulated in MHCC97-H

thus the results of Western blot were consistent with those of LC-MS/MS. Conclusion:PLEC and SHMT2 are validated to be novel candidate HCC metastasis-associated proteins based on the analysis of this study.