High-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry, two-dimensional difference in-gel electrophoresis and gene microarray identification of lymphatic metastasis-associated biomarkers.

The potential biomarkers for the lymphatic metastatic process of mouse hepatocarcinoma were investigated by using two-dimensional difference in-gel electrophoresis (2D DIGE), high-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry (HPLC/nESI-MS/MS) and GeneChip. 2D DIGE was performed to screen and quantify the differentially expressed proteins between two well-established mouse hepatocarcinoma cell lines, Hca-F with 75% and Hca-P with 25% metastasis rate of lymph node potentials. The protein spots in the gel were visualized by the highly sensitive Deep Purple (GE Healthcare) fluorescent stain. Protein identification was obtained for gel spots by HPLC/nESI-MS/MS analysis with high quality. GeneChip microarray was performed to identify genes differentially expressed at the mRNA level. Seventeen genes including the chloride intracellular channel l, caspase 3, fructose bisphosphatase 2, glutamate dehydrogenase 1, V-crk sarcoma virus CT10 oncogene homolog, N-myc downstream regulated gene1, villin2, gelsolin, enoyl coenzyme A hydratase 1, transketolase, vimentin, annexins A5 and A7, keratin complex2 basic gene7 and gene8, lactamase (bata 2) and Ero1-like protein were found abnormally regulated and expressed concordantly both at the protein and mRNA levels between the two cell lines. More than half of these genes were for the first time revealed to be involved directly in hepatocarcinoma due to the lymphatic metastasis. The interdisciplinary combination of HPLC/nESI-MS/MS with 2D DIGE and GeneChip techniques opens up the possibility for the biomarker discovery of disease with high confidence.