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High throughput screening of disulfide-containing proteins in a complex mixture

Authors

  • Dong S. Zhao,

    1. The Human Proteomics Program, University of Wisconsin-Madison, Madison, WI, USA
    Current affiliation:
    1. Irvine Pharmaceutical Services, Inc., Irvine, CA 92618
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  • Zachery R. Gregorich,

    1. The Human Proteomics Program, University of Wisconsin-Madison, Madison, WI, USA
    2. Molecular and Cellular Pharmacology Training Program, University of Wisconsin-Madison, Madison, WI, USA
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  • Ying Ge

    Corresponding author
    1. The Human Proteomics Program, University of Wisconsin-Madison, Madison, WI, USA
    2. Molecular and Cellular Pharmacology Training Program, University of Wisconsin-Madison, Madison, WI, USA
    3. Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA
    4. Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA
    • Correspondence: Professor Ying Ge, University of Wisconsin-Madison, 1300 University Avenue, SMI 130, Madison, WI 53706, USA

      E-mail: ge2@wisc.edu

      Fax: +1 608-265-5512

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Abstract

The formation of disulfide bonds between cysteine residues is crucial for the stabilization of native protein structures and, thus, determination of disulfide linkages is an important facet of protein structural characterization. Nonetheless, the identification of disulfide bond linkages remains a significant analytical challenge, particularly in large proteins with complex disulfide patterns. Herein, we have developed a new LC/MS strategy for rapid screening of disulfides in an intact protein mixture after a straightforward reduction step with tris(2-carboxyethyl)phosphine. LC/MS analysis of reduced and nonreduced protein mixtures quickly revealed disulfide-containing proteins owing to a 2 Da mass increase per disulfide reduction and, subsequently, the total number of disulfide bonds in the intact proteins could be determined. We have demonstrated the effectiveness of this method in a protein mixture composed of both disulfide-containing and disulfide-free proteins. Our method is simple (no need for proteolytic digestion, alkylation, or the removal of reducing agents prior to MS analysis), high throughput (fast on-line LC/MS analysis), and reliable (no S–S scrambling), underscoring its potential as a rapid disulfide screening method for proteomics applications.

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