A lab-made off line 2D-LC-MS system was developed for protein analysis. In case the off line 2D-LC was utilized for peptide separation, and the working conditions for strong cation exchange(SCX) chromatography and the succeeding reversed phase(RP) chromatography can be individually optimized, contrasted to the on-line system. The experimental results show that the mass fractions of acetonitrile for the SCX separation can be increased form 5% as references reported up to 20% so that the separation of peptides has been substantially improved. The off line system has been applied to the analysis of proteins extracted from the cancer tissue of nude mice inoculated by a human-hepatocellular-carcinoma cell-line D20. Some extreme basic proteins(pI>10) and larger proteins(M w>200 000), which are difficult to be isolated by the traditional 2D-PAGE method, have been identified in D20 sample.
