Abstract
A method for characterizing and quantifying peaks formed in an analytical buoyant density equilibrium (ABDE) experiment is presented. An algorithm is derived to calculate the concentration of the density forming gradient material at every point in the cell, provided the rotor speed, temperature, meniscus position, bottom of the cell position, and the loading concentration, molar mass, and partial specific volume of the density gradient-forming material are known. In addition, a new peak fitting algorithm has been developed which allows the user to automatically quantify the peaks formed in terms of density, apparent partial specific volume, and relative abundance. The method is suitable for both ionic and non-ionic density forming materials and can be used with data generated from the UV optical system as well as the AVIV fluorescence optical system. These methods have been programmed in a new UltraScan-III module (us_abde). Examples are shown that demonstrate the application of the new module to adeno-associated viral vector preparations and proteins.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The UltraScan software used to analyze the AUC data is open source and freely available from the Github repository (https://github.com/ehb54/ultrascan3). The AUC data is available in openAUC format upon request from the authors, and is stored in the UltraScan LIMS server at the Canadian Center for Hydrodynamics.
References
Challis RC, Ravindra Kumar S, Chan KY, Challis C, Beadle K, Jang MJ, Kim HM, Rajendran PS, Tompkins JD, Shivkumar K, Deverman BE, Gradinaru V (2019) Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat Protoc 14(2):379–414. https://doi.org/10.1038/s41596-018-0097-3
Cölfen H, Laue TM, Wohlleben W, Schilling K, Karabudak E, Langhorst BW, Brookes E, Dubbs B, Zollars D, Rocco M, Demeler B (2010) The open AUC project. Eur Biophys J 39(3):347–359. https://doi.org/10.1007/s00249-009-0438-9
Demeler B (2005) UltraScan a comprehensive data analysis software package for analytical ultracentrifugation experiments. In: Scott DJ, Harding SE, Rowe AJ (eds) Modern analytical ultracentrifugation: techniques and methods. Royal Society of Chemistry (UK), p 210–229
Demeler B (2011) UltraScan-II version 9.9, release 1585-a comprehensive data analysis software package for analytical ultracentrifugation experiments. https://github.com/ehb54/ultrascan2
Demeler B, van Holde KE (2004) Sedimentation velocity analysis of highly heterogeneous systems. Anal Biochem 335(2):279–288. https://doi.org/10.1016/j.ab.2004.08.039
Demeler B, Brookes EH (2008) Monte Carlo analysis of sedimentation experiments. Colloid Polym Sci 286:129–137
Demeler B, Gorbet G (2016) Analytical ultracentrifugation data analysis with UltraScan-III. Ch. 8. In: Uchiyama S, Stafford WF, Laue T (eds) Analytical ultracentrifugation: instrumentation, software, and applications. Springer, Japan, pp 119–143
Demeler B, Brookes EH, Gorbet GE, Savelyev A, Zollars D, Dubbs B (2022) UltraScan data analysis software for analytical ultracentrifugation experiments and hydrodynamic modeling. https://github.com/ehb54/ultrascan3
Deng S, Oka K (2020) Adeno-associated virus as gene delivery vehicle into the retina. Methods Mol Biol 2092:77–90
Green AA, Hersam MC (2011) Nearly single-chirality single-walled carbon nanotubes produced via orthogonal iterative density gradient ultracentrifugation. Adv Mater 23(19):2185–2190. https://doi.org/10.1002/adma.201100034
Henrickson A, Gorbet GE, Savelyev A, Kim M, Hargreaves J, Schultz SK, Kothe U, Demeler B (2022) Multi-wavelength analytical ultracentrifugation of biopolymer mixtures and interactions. Anal Biochem 652:114728. https://doi.org/10.1016/j.ab.2022.114728
MacGregor IK, Anderson AL, Laue TM (2004) Fluorescence detection for the XLI analytical ultracentrifuge. Biophys Chem 108:165–185
Memon S, Riedel M, Janetzko F, Demeler B, Gorbet G, Marru S, Grimshaw A, Gunathilake L, Singh R, AttigLippert NT (2014) Advancements of the UltraScan scientific gateway for open standards-based cyberinfrastructures. Concurr Comput Pract Exper. https://doi.org/10.1002/cpe.3251
Meselson M, Stahl FW (1958) The replication of DNA in Escherichia coli. Proc Natl Acad Sci 44:671–682
Mortezazadeh S, Demeler B (2023) Systematic noise removal from analytical ultracentrifugation data with UltraScan. Eur Biophys J. https://doi.org/10.1007/s00249-023-01631-6
Sambrook J, Fritsch ER, Maniatis T (1989) Molecular cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Stoutjesdyk M, Henrickson A, Minors G, Demeler B (2020) A calibration disk for the correction of radial errors from chromatic aberration and rotor stretch in the Optima AUC™ analytical ultracentrifuge. Eur Biophys J 49(8):701–709. https://doi.org/10.1007/s00249-020-01434-z
The Axis-Shield Product information guide for Nycodenz®. https://www.proteogenix-products.com/documents/pdf/axis-shield/1002424-Nycodenz.pdf
Wang S, Guo Y, Pu WT (2021) AAV gene transfer to the heart. Methods Mol Biol 2158:269–280. https://doi.org/10.1007/978-1-0716-0668-1_20
Acknowledgements
We thank Viviana Gradinaru and Zhe Qu, California Institute of Technology, for providing the AAV9 sample, and thank Saeed Mortezazadeh, University of Lethbridge for assistance with the pseudo-absorbance module in UltraScan. We also thank Lauren Tsai and Micah Tatum, The University of Texas Health Science Center San Antonio, for performing the Nycodenz measurements. This work was supported by the Biomolecular Interaction Technology Center, University of Delaware, the Canada 150 Research Chairs program (C150-2017-00015, BD), the Canada Foundation for Innovation (CFI-37589, BD), the National Institutes of Health (1R01GM120600 to EB, AS, BD) and the Canadian Natural Science and Engineering Research Council (DG-RGPIN-2019-05637, BD). The Canadian Natural Science and Engineering Research Council is acknowledged for supporting AH through a scholarship grant.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Special Issue: Analytical Ultracentrifugation 2022.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Savelyev, A., Brookes, E.H., Henrickson, A. et al. A new UltraScan module for the characterization and quantification of analytical buoyant density equilibrium experiments to determine AAV capsid loading. Eur Biophys J 52, 311–320 (2023). https://doi.org/10.1007/s00249-023-01641-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-023-01641-4