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Selective Comprehensive Online NanoLCxCZE-MS Platform for Top-Down Proteoform Analysis.

Tobias Waldmann1, Philipp T Kaulich2, Alexander Stolz1

  • 1Faculty of Chemistry, Aalen University, Aalen 73430, Germany.

Analytical Chemistry
|October 17, 2025
PubMed
Summary

This study introduces a novel 2D separation platform combining nanoLC and capillary zone electrophoresis-mass spectrometry (CZE-MS) for enhanced proteoform analysis. The new method significantly increases the detection of intact proteoforms in complex biological samples.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Efficient separation techniques are essential for comprehensive proteoform analysis in complex biological samples.
  • Two-dimensional (2D) separation methods offer superior selectivity and peak capacity over one-dimensional (1D) approaches.

Purpose of the Study:

  • To develop and present a novel online selective comprehensive 2D platform coupling reversed-phase nanoLC with capillary zone electrophoresis-mass spectrometry (CZE-MS).
  • To apply this platform for intact top-down (TD) proteoform analysis.
  • To enhance the detection and characterization of proteoforms in complex biological samples.

Main Methods:

  • Development of an online selective comprehensive 2D platform integrating reversed-phase nanoLC (1st dimension) and capillary zone electrophoresis-mass spectrometry (CZE-MS) (2nd dimension).
  • Utilized successive multiple ionic-polymer layers for enhanced CZE-MS separation.
  • Analyzed a human cell lysate to evaluate the platform's performance for intact TD proteoform characterization.

Main Results:

  • The 2D platform detected two to four times more proteoforms compared to 1D nanoLC-MS within the same nanoLC time window.
  • Targeted analysis of Histone H4 identified additional proteoforms with variations in acetylation and methylation previously undetected by 1D nanoLC-MS.
  • Demonstrated the platform's capability for advanced intact proteoform analysis.

Conclusions:

  • The coupled nanoLC and CZE-MS platform provides significantly improved proteoform detection and characterization at the intact protein level.
  • This 2D approach advances targeted proteoform analysis in complex biological matrices.
  • The developed platform holds promise for future in-depth proteomic studies.