Ultrahigh pressure fast size exclusion chromatography for top-down proteomics

Authors

  • Xin Chen,

    1. Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA
    2. Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA
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  • Ying Ge

    Corresponding author
    1. Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA
    2. Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA
    • Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA
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  • Colour Online: See the article online to view Figs. 1–3 in colour.

Correspondence: Dr. Ying Ge, Department of Cell and Regenerative Biology, University of Wisconsin-Madison, 1300 University Avenue, SMI 130, Madison, WI 53706, USA

E-mail: ge2@wisc.edu

Fax: +1 608-265-5512

Abstract

Top-down MS-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6–669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC-eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size separation of proteins with great potential for top-down proteomics.

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