High Performance Liquid Chromatography (HPLC)

Although two-dimensional gel electrophoresis (2-DE) is a commonly used method for analyzing the whole proteome, it has limitations such as limited separation ability, discriminatory effect, and complicated operating procedures. It is often difficult to obtain satisfactory results for the analysis of large dynamic range, low abundance and hydrophobic proteins. Chong et al. used HPLC/mass spectrometry to compare and analyze the differential expression of pre-malignant and cancer proteins. The protein was separated by HPLC, and the collected fractions were identified by MALDI-TOF-MS, so that the protein was quantitatively analyzed in the differential expression of the two kinds of cells. As a new type of separation technology, multidimensional liquid chromatography does not have the limitation of relative molecular mass and isoelectric point. Through the combination of different modes, it eliminates the discriminatory effect of two-dimensional gel electrophoresis. It has the characteristics of high peak capacity and easy automation. Two-dimensional ion exchange-reverse phase chromatography (2D-IEC-RPLC) is the most commonly used multi-dimensional liquid chromatography separation system in proteomics research.