High-pH Reversed-Phase Pre-Fractionation for In-Depth Shotgun Proteomics

  • Ning Chen
  • Mingwei Liu
  • Jun Qin
  • Wei SunEmail author
  • Fuchu HeEmail author
Part of the Neuromethods book series (NM, volume 146)


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.


High pH Reverse phase Fractionation Liquid chromatography Two-dimensional chromatography Mass spectrometry Proteomics 


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences BeijingBeijing Institute of LifeomicsBeijingChina

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