Characterizing non-covalent protein complexes using asymmetrical flow field-flow fractionation on-line coupled to native mass spectrometry
- Author
- Iro Konstantina Ventouri, Wayne Chang, Florian Meier, Roland Drexel, Govert W. Somsen, Peter J. Schoenmakers, Bart De Spiegeleer (UGent) , Rob Haselberg and Alina Astefanei
- Organization
- Abstract
- We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV absorbance, multi-angle light scattering (MALS), and differential refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS- dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) L-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4- nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.
- Keywords
- SIZE-EXCLUSION CHROMATOGRAPHY, L-ASPARAGINASE, LIQUID-CHROMATOGRAPHY, TOP-DOWN
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-01HMRNSN9Q82PA7GF05RXES8CR
- MLA
- Ventouri, Iro Konstantina, et al. “Characterizing Non-Covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation on-Line Coupled to Native Mass Spectrometry.” ANALYTICAL CHEMISTRY, vol. 95, no. 19, 2023, pp. 7487–94, doi:10.1021/acs.analchem.2c05049.
- APA
- Ventouri, I. K., Chang, W., Meier, F., Drexel, R., Somsen, G. W., Schoenmakers, P. J., … Astefanei, A. (2023). Characterizing non-covalent protein complexes using asymmetrical flow field-flow fractionation on-line coupled to native mass spectrometry. ANALYTICAL CHEMISTRY, 95(19), 7487–7494. https://doi.org/10.1021/acs.analchem.2c05049
- Chicago author-date
- Ventouri, Iro Konstantina, Wayne Chang, Florian Meier, Roland Drexel, Govert W. Somsen, Peter J. Schoenmakers, Bart De Spiegeleer, Rob Haselberg, and Alina Astefanei. 2023. “Characterizing Non-Covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation on-Line Coupled to Native Mass Spectrometry.” ANALYTICAL CHEMISTRY 95 (19): 7487–94. https://doi.org/10.1021/acs.analchem.2c05049.
- Chicago author-date (all authors)
- Ventouri, Iro Konstantina, Wayne Chang, Florian Meier, Roland Drexel, Govert W. Somsen, Peter J. Schoenmakers, Bart De Spiegeleer, Rob Haselberg, and Alina Astefanei. 2023. “Characterizing Non-Covalent Protein Complexes Using Asymmetrical Flow Field-Flow Fractionation on-Line Coupled to Native Mass Spectrometry.” ANALYTICAL CHEMISTRY 95 (19): 7487–7494. doi:10.1021/acs.analchem.2c05049.
- Vancouver
- 1.Ventouri IK, Chang W, Meier F, Drexel R, Somsen GW, Schoenmakers PJ, et al. Characterizing non-covalent protein complexes using asymmetrical flow field-flow fractionation on-line coupled to native mass spectrometry. ANALYTICAL CHEMISTRY. 2023;95(19):7487–94.
- IEEE
- [1]I. K. Ventouri et al., “Characterizing non-covalent protein complexes using asymmetrical flow field-flow fractionation on-line coupled to native mass spectrometry,” ANALYTICAL CHEMISTRY, vol. 95, no. 19, pp. 7487–7494, 2023.
@article{01HMRNSN9Q82PA7GF05RXES8CR, abstract = {{We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV absorbance, multi-angle light scattering (MALS), and differential refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS- dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) L-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4- nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.}}, author = {{Ventouri, Iro Konstantina and Chang, Wayne and Meier, Florian and Drexel, Roland and Somsen, Govert W. and Schoenmakers, Peter J. and De Spiegeleer, Bart and Haselberg, Rob and Astefanei, Alina}}, issn = {{0003-2700}}, journal = {{ANALYTICAL CHEMISTRY}}, keywords = {{SIZE-EXCLUSION CHROMATOGRAPHY,L-ASPARAGINASE,LIQUID-CHROMATOGRAPHY,TOP-DOWN}}, language = {{eng}}, number = {{19}}, pages = {{7487--7494}}, title = {{Characterizing non-covalent protein complexes using asymmetrical flow field-flow fractionation on-line coupled to native mass spectrometry}}, url = {{http://doi.org/10.1021/acs.analchem.2c05049}}, volume = {{95}}, year = {{2023}}, }
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