Chinese Journal of Chromatography ›› 2019, Vol. 37 ›› Issue (5): 477-483.DOI: 10.3724/SP.J.1123.2018.11021

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Establishment of a microscale peptide prefractionation system based on ultra performance liquid chromatography and the eight-port rotor valve

YAN Meng1, ZHAO Yang2, ZHANG Yangjun2, YING Wantao2, QIAN Xiaohong1,2   

  1. 1. College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China;
    2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences(Beijing), Beijing Institute of Lifeomics, Beijing 102206, China
  • Received:2018-11-19 Online:2019-05-08 Published:2015-03-23
  • Supported by:

    National Key Program for Basic Research of China (Nos. 2017YFC0906600, 2016YFA0501300); National Natural Science Foundation of China (No. 81530021); Innovation Project of Military Medicine (No. 16CXZ027).

Abstract:

Liquid chromatography (LC) and mass spectrometry (MS)-based proteomics now allows very deep coverage of proteomes. Two-dimensional high performance liquid chromatography (2-D HPLC) is a useful tool for the proteome analysis of complex biosystem. However, it has drawback of a long running time, and it typically requires peptide amounts in the milligram range, and large volume of collected fractions. In this study, we introduce ultra-performance liquid chromatography (UPLC) and an eight-port rotor valve as a highly efficient and convenient method for a first-dimension separation and collection system. The combination of our UPLC-based fractionation using basic buffers with an online LC-MS/MS provided orthogonal peptide separation and demonstrated the powerful performance for protein identification. Upon applying the novel method to triplicate measurements of a human cell line, we observed excellent quantitative reproducibility between replicates (coefficient of determination R2>0.95) and more than 23.52% peptide identifications over the conventional StageTip approach. The fractionation method described here is flexible, straightforward, and robust, and it enables proteome analysis with minimal sample requirements.

Key words: fractionation, proteomics, the eight-port rotor valve, two-dimensional liquid chromatography (2-D LC), ultra performance liquid chromatography (UPLC)

CLC Number: