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