Differential gel-based proteomic approach for cancer biomarker discovery using human plasma

Two-dimensional fluorescence difference gel electrophoresis (2D DIGE) has become a general platform for analysis of various clinical samples such as biofluids and tissues. In comparison to conventional 2-D polyacrylamide gel electrophoresis (2D PAGE), 2D DIGE offers several advantages, such as accuracy and reproducibility between experiments, which facilitate spot-to-spot comparisons. Although whole plasma can be easily obtained, the complexity of plasma samples makes it challenging to analyze samples with good reproducibility. Here, we describe a method for decreasing protein complexity in plasma samples within a narrow pH range by depleting high-abundance plasma proteins. In combination with analysis of differentially expressed spots, trypsin digestion, identification of protein by mass spectrometry, and standard 2D PAGE and DIGE, this method has been optimized for comparison of plasma samples from healthy donors and patients diagnosed with hepatocellular carcinoma.