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Related Experiment Video

Updated: Sep 21, 2025

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry
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Membrane Ultrafiltration-Based Sample Preparation Method and Sheath-Flow CZE-MS/MS for Top-Down Proteomics.

Zhichang Yang1, Liangliang Sun2

  • 1Department of Chemistry, Michigan State University, East Lansing, MI, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 3, 2022
PubMed
Summary

A new membrane ultrafiltration (MU) method efficiently removes detergents for mass spectrometry (MS)-based denaturing top-down proteomics (dTDP). This technique enables robust proteoform characterization, identifying over 800 proteoforms in E. coli.

Keywords:
Capillary zone electrophoresis-mass spectrometryDenaturing top-down proteomicsMass spectrometryMembrane ultrafiltrationProteoformSodium dodecyl sulfate

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) enables proteoform identification without enzymatic digestion.
  • Effective sample preparation is crucial for large-scale proteoform characterization, requiring efficient protein extraction, minimal loss, solubility maintenance, and compatibility with liquid-phase separation and MS/MS.

Purpose of the Study:

  • To develop a universal sample preparation method for dTDP.
  • To optimize a method compatible with liquid-phase separation and MS/MS analysis for comprehensive proteoform characterization.

Main Methods:

  • Membrane ultrafiltration (MU) was utilized for buffer exchange to remove sodium dodecyl sulfate (SDS).
  • Protein samples were prepared using MU followed by capillary zone electrophoresis-tandem MS (CZE-MS/MS) analysis.
  • The method was applied to an Escherichia coli (E. coli) sample.

Main Results:

  • The MU method demonstrated efficient detergent removal and good protein recovery.
  • The technique exhibited minimal protein bias and excellent compatibility with CZE-MS/MS.
  • Single-shot CZE-MS/MS analysis identified over 800 proteoforms in the E. coli sample.

Conclusions:

  • Membrane ultrafiltration provides an efficient and compatible sample preparation strategy for MS-based denaturing top-down proteomics.
  • This method facilitates large-scale proteoform characterization with high throughput and minimal sample loss.
  • The developed MU-CZE-MS/MS workflow significantly advances the capabilities for analyzing complex proteomes.