High-pH Reversed-Phase Pre-Fractionation for In-Depth Shotgun Proteomics
Two-dimensional LC-MS-based proteomics is a powerful strategy for high-throughput protein identification and quantification. The combination of high-pH reversed-phase fractionation with low-pH LC-MS analysis is the method of choice, because it provides the optimal separation efficiency and deep protein coverage. In this chapter, we illustrate the protocols of HPLC-based and the tip-based high-pH reversed-phase chromatographic fractionation of peptide mixtures, in which the HPLC-based reversed-phase fractionation provides higher resolving power while the tip-based high-pH reversed-phase fractionation is simple, parallelizable, and suitable for limited amount of biological samples.
KeywordsHigh pH Reverse phase Fractionation Liquid chromatography Two-dimensional chromatography Mass spectrometry Proteomics