Roemer, Jennifer and Montealegre, Cristina and Schlecht, Johannes and Kiessig, Steffen and Moritz, Bernd and Neusuess, Christian (2019) Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 411 (27). pp. 7197-7206. ISSN 1618-2642, 1618-2650
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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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | SODIUM DODECYL-SULFATE; ACID-LABILE SURFACTANT; MONOCLONAL-ANTIBODY; GEL-ELECTROPHORESIS; SEPARATION SYSTEMS; QUALITY-CONTROL; IDENTIFICATION; HETEROGENEITY; DISULFIDE; LIQUID; Capillary electrophoresis; Two-dimension; CE (SDS); Monoclonal antibody; Sodium dodecyl sulfate; Mass spectrometry |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Institut für Analytische Chemie, Chemo- und Biosensorik |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 25 Mar 2020 10:13 |
| Last Modified: | 25 Mar 2020 10:13 |
| URI: | https://pred.uni-regensburg.de/id/eprint/26002 |
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