Analytical and Bioanalytical Chemistry

, Volume 411, Issue 27, pp 7197–7206 | Cite as

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis

  • Jennifer Römer
  • Cristina Montealegre
  • Johannes Schlecht
  • Steffen Kiessig
  • Bernd Moritz
  • Christian NeusüßEmail author
Research Paper


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20 nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization.

Graphical abstract

Two-dimensional capillary electrophoresis system hyphenated with mass spectrometry for the characterization of CE (SDS)-separated proteins. As first dimension, a generic and high MS-interfering CE (SDS) separation is performed for size separation. After heart-cut transfer of the unknown CE (SDS) protein peak, via a four-port nanoliter valve to a volatile electrolyte system as second dimension, interference-free mass spectrometric data of separated mAb fragments and soybean proteins are obtained.


Capillary electrophoresis Two-dimension CE (SDS) Monoclonal antibody Sodium dodecyl sulfate Mass spectrometry 



The authors gratefully acknowledge Dr. Laura Sánchez-Hernández for helpful discussions and Kevin Jooß for the support in data interpretation.

Author’s contributions

JR, CM, and JS developed the methods and performed experiments, analyzed data, and wrote the paper. SK and BM conceived the approach and wrote the paper. CN initiated and supervised the project, conceived the approach, and wrote the paper.

Funding information

This study was funded by F. Hoffmann-La Roche Ltd.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_2102_MOESM1_ESM.pdf (440 kb)
ESM 1 (PDF 439 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jennifer Römer
    • 1
    • 2
  • Cristina Montealegre
    • 1
  • Johannes Schlecht
    • 1
    • 3
  • Steffen Kiessig
    • 3
  • Bernd Moritz
    • 3
  • Christian Neusüß
    • 1
    Email author
  1. 1.Faculty of ChemistryAalen UniversityAalenGermany
  2. 2.Institute of Analytical Chemistry, Chemo- and BiosensorsUniversity of RegensburgRegensburgGermany
  3. 3.F. Hoffmann-La Roche LtdBaselSwitzerland

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