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An automatic system with sample introduction and multi-dimensional separation for nanoflow liquid chromatography-tandem mass spectrometric (μLC-MS/MS) analysis was developed by combination of a strong cation exchange (SCX) trap column with a reversed-phase separation column.During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading large volume of sample at high flow rate.Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed with RP chromatographic separation of the peptides at nanoliter flow rate.