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Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture
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Fast and Accurate Disulfide Bridge Detection.

Søren Heissel1, Yi He2, Andris Jankevics3

  • 1Proteomics Resource Center, The Rockefeller University, New York, New York, USA.

Molecular & Cellular Proteomics : MCP
|April 4, 2024
PubMed
Summary

Accurate disulfide bridge mapping in proteins is crucial for quality control. This study introduces a rapid, 1-hour workflow using mass spectrometry for high-throughput, high-accuracy assessment of these critical protein structures.

Keywords:
EThcDFAIMSMAAHXlinkX/PDdisulfide bridge

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Area of Science:

  • Biochemistry and Proteomics
  • Protein Engineering and Quality Control

Background:

  • Recombinant protein production is a major industry, necessitating robust quality control for attributes like sequence fidelity, folding, and post-translational modifications.
  • Disulfide bridges are critical for protein folding, stability, and bioactivity, but their accurate assessment has been challenging.
  • Errors in protein attributes can lead to reduced efficacy and increased immunogenicity risk for therapeutic proteins.

Purpose of the Study:

  • To develop a rapid and accurate method for assessing disulfide bridges in purified proteins.
  • To establish a high-throughput workflow for disulfide mapping in protein quality control.

Main Methods:

  • Utilized microwave-assisted acid hydrolysis for rapid, artifact-free protein digestion into overlapping peptides.
  • Integrated ion mobility spectrometry to remove chemical background noise from nonspecific digestion.
  • Extended the XlinkX node in Proteome Discoverer for efficient data analysis and false discovery rate correction.

Main Results:

  • Developed a unique workflow combining sample preparation, data acquisition, and analysis for disulfide bridge assessment.
  • Achieved rapid and accurate assessment of disulfide bridges in purified proteins.
  • The entire workflow can be completed within one hour, enabling high-throughput analysis.

Conclusions:

  • The presented workflow enables fast and accurate disulfide mapping, addressing a key challenge in protein quality control.
  • This method facilitates high-throughput assessment, crucial for the large-scale production of therapeutic proteins.
  • The optimized workflow enhances the reliability of protein characterization, ensuring bioactivity and minimizing immunogenicity risks.