A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Langowski, J. and Zhao, H. and Ghirlando, R. and Alfonso, C. and Arisaka, F. and Attali, I. and Bain, D.L. and Bakhtina, M.M. and Becker, D.F. and Bedwell, G.J. and Bekdemir, A. and Besong, T.M.D. and Birck, C. and Brautigam, C.A. and Brennerman, W. and Byron, O. and Bzowska, A. and Chaires, J.B. and Chaton, C.T. and Cölfen, H. and Connaghan, K.D. and Crowley, K.A. and Curth, U. and Daviter, Tina and Dean, W.L. and Díez, A.I. and Ebel, C. and Eckert, D.M. and Eisele, L.E. and Eisenstein, E. and England, P. and Escalante, C. and Fagan, J.A. and Fairman, R. and Finn, R.M. and Fischle, W. and de la Torre, J.G. and Gor, J. and Gustafsson, H. and Hall, D. and Harding, S.E. and Cifre, J.G.H. and Herr, A.B. and Howell, E.E. and Isaac, R.S. and Jao, S.-C. and Jose, D. and Kim, S.-J. and Kokona, B. and Kornblatt, J.A. and Kosek, D. and Krayukhina, E. and Krzizike, D. and Kusznir, E.A. and Kwon, H. and Larson, A. and Laue, T.M. and Le Roy, A. and Leech, A.P. and Lilie, H. and Luger, K. and Luque-Ortega, J.R. and Ma, J. and May, C.A. and Maynard, E.L. and Modrak-Wojcik, A. and Mok, Y.-F. and Mücke, N. and Nagel-Steger, L. and Narlikar, G.J. and Noda, M. and Nourse, A. and Obsil, T. and Park, C.K. and Park, J.-K. and Pawelek, P.D. and Perdue, E.E. and Perkins, S.J. and Perugini, M.A. and Peterson, C.L. and Peverelli, M.G. and Piszczek, G. and Prag, G. and Prevelige, P.E. and Raynal, B.D.E. and Rezabkova, L. and Richter, K. and Ringel, A.E. and Rosenberg, R. and Rowe, A.J. and Rufer, A.C. and Scott, D.J. and Seravalli, J.G. and Solovyova, A.S. and Song, R. and Staunton, D. and Stoddard, C. and Stott, K. and Strauss, H.M. and Streicher, W.W. and Sumida, J.P. and Swygert, S.G. and Szczepanowski, R.H. and Tessmer, I. and Toth, R.T. and Tripathy, A. and Uchiyama, S. and Uebel, S.F.W. and Unzai, S. and Gruber, A.V. and von Hippel, P.H. and Wandrey, C. and Wang, S.-H. and Weitzel, S.E. and Wielgus-Kutrowska, B. and Wolberger, C. and Wolff, M. and Wright, E. and Wu, Y.-S. and Wubben, J.M. and Schuck, P. (2015) A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PLoS One 10 (5), e0126420. ISSN 1932-6203.

Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

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