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

Zhao H, Ghirlando R, Alfonso C, Arisaka F, Attali I, Bain DL, Bakhtina MM, Becker DF, Bedwell GJ, Bekdemir A, Besong TM, Birck C, Brautigam CA, Brennerman W, Byron O, Bzowska A, Chaires JB, Chaton CT, Cölfen H, Connaghan KD, Crowley KA, Curth U, Daviter T, Dean WL, Díez AI, Ebel C, Eckert DM, Eisele LE, Eisenstein E, England P, Escalante C, Fagan JA, Fairman R, Finn RM, Fischle W, de la Torre JG, Gor J, Gustafsson H, Hall D, Harding SE, Cifre JG, Herr AB, Howell EE, Isaac RS, Jao SC, Jose D, Kim SJ, Kokona B, Kornblatt JA, Kosek D, Krayukhina E, Krzizike D, Kusznir EA, Kwon H, Larson A, Laue TM, Le Roy A, Leech AP, Lilie H, Luger K, Luque-Ortega JR, Ma J, May CA, Maynard EL, Modrak-Wojcik A, Mok YF, Mücke N, Nagel-Steger L, Narlikar GJ, Noda M, Nourse A, Obsil T, Park CK, Park JK, Pawelek PD, Perdue EE, Perkins SJ, Perugini MA, Peterson CL, Peverelli MG, Piszczek G, Prag G, Prevelige PE, Raynal BD, Rezabkova L, Richter K, Ringel AE, Rosenberg R, Rowe AJ, Rufer AC, Scott DJ, Seravalli JG, Solovyova AS, Song R, Staunton D, Stoddard C, Stott K, Strauss HM, Streicher WW, Sumida JP, Swygert SG, Szczepanowski RH, Tessmer I, Toth RT, Tripathy A, Uchiyama S, Uebel SF, Unzai S, Gruber AV, von Hippel PH, Wandrey C, Wang SH, Weitzel SE, Wielgus-Kutrowska B, Wolberger C, Wolff M, Wright E, Wu YS, Wubben JM, Schuck P - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, 20892, United States of America.

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.

No MeSH data available.


Root-mean-square deviation of the best-fit c(s) model of the BSA sedimentation experiment when scanned with the absorbance system (green) and the interference system (magenta).The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.
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pone.0126420.g008: Root-mean-square deviation of the best-fit c(s) model of the BSA sedimentation experiment when scanned with the absorbance system (green) and the interference system (magenta).The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.

Mentions: The design of the study was such that the analysis of the sedimentation boundaries recorded from the BSA sample would allow for an assessment of the completeness and internal consistency of the external calibration corrections in time, temperature, and radial magnification. Generally the quality of fit of the experimental SV data was excellent (for a representative BSA sedimentation velocity absorbance analysis, see Fig 7), with an average rmsd of 0.0067 OD in the absorbance system and 0.0061 fringes in the interference system. However, as shown in Fig 8, the distribution of rmsd values showed a minority of outliers. Some of the outliers in the absorbance data set can be traced simply to very noisy data resulting from low signal intensities below 800 counts. However, at 800 counts and above, no correlation between the rmsd of the fit and reported signal intensity counts was found (data not shown). In other cases, the rmsd was particularly high in the initial scans close to the meniscus region, pointing to convection as an origin of the misfit (e.g., S7 Fig). Interestingly, in a few instruments, the interference data exhibited an unexplained pattern of sloping plateaus (e.g., S8 Fig), which required truncation of the radial range used for analysis and correlated with increased errors in the determination of boundary magnitudes (data not shown). Finally, a few interference data sets exhibited wavy patterns superimposed onto the boundary shapes, but without causing excessively high rmsd or outlier s- and M-values (data not shown). Overall, in 11 out of 119 experiments the data acquisition was either not attempted, failed, or produced data sets where the c(s) model did not lead to baseline separation between the BSA monomer and dimer peak; these data sets were not included in the analysis of s-values.


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

Zhao H, Ghirlando R, Alfonso C, Arisaka F, Attali I, Bain DL, Bakhtina MM, Becker DF, Bedwell GJ, Bekdemir A, Besong TM, Birck C, Brautigam CA, Brennerman W, Byron O, Bzowska A, Chaires JB, Chaton CT, Cölfen H, Connaghan KD, Crowley KA, Curth U, Daviter T, Dean WL, Díez AI, Ebel C, Eckert DM, Eisele LE, Eisenstein E, England P, Escalante C, Fagan JA, Fairman R, Finn RM, Fischle W, de la Torre JG, Gor J, Gustafsson H, Hall D, Harding SE, Cifre JG, Herr AB, Howell EE, Isaac RS, Jao SC, Jose D, Kim SJ, Kokona B, Kornblatt JA, Kosek D, Krayukhina E, Krzizike D, Kusznir EA, Kwon H, Larson A, Laue TM, Le Roy A, Leech AP, Lilie H, Luger K, Luque-Ortega JR, Ma J, May CA, Maynard EL, Modrak-Wojcik A, Mok YF, Mücke N, Nagel-Steger L, Narlikar GJ, Noda M, Nourse A, Obsil T, Park CK, Park JK, Pawelek PD, Perdue EE, Perkins SJ, Perugini MA, Peterson CL, Peverelli MG, Piszczek G, Prag G, Prevelige PE, Raynal BD, Rezabkova L, Richter K, Ringel AE, Rosenberg R, Rowe AJ, Rufer AC, Scott DJ, Seravalli JG, Solovyova AS, Song R, Staunton D, Stoddard C, Stott K, Strauss HM, Streicher WW, Sumida JP, Swygert SG, Szczepanowski RH, Tessmer I, Toth RT, Tripathy A, Uchiyama S, Uebel SF, Unzai S, Gruber AV, von Hippel PH, Wandrey C, Wang SH, Weitzel SE, Wielgus-Kutrowska B, Wolberger C, Wolff M, Wright E, Wu YS, Wubben JM, Schuck P - PLoS ONE (2015)

Root-mean-square deviation of the best-fit c(s) model of the BSA sedimentation experiment when scanned with the absorbance system (green) and the interference system (magenta).The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440767&req=5

pone.0126420.g008: Root-mean-square deviation of the best-fit c(s) model of the BSA sedimentation experiment when scanned with the absorbance system (green) and the interference system (magenta).The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.
Mentions: The design of the study was such that the analysis of the sedimentation boundaries recorded from the BSA sample would allow for an assessment of the completeness and internal consistency of the external calibration corrections in time, temperature, and radial magnification. Generally the quality of fit of the experimental SV data was excellent (for a representative BSA sedimentation velocity absorbance analysis, see Fig 7), with an average rmsd of 0.0067 OD in the absorbance system and 0.0061 fringes in the interference system. However, as shown in Fig 8, the distribution of rmsd values showed a minority of outliers. Some of the outliers in the absorbance data set can be traced simply to very noisy data resulting from low signal intensities below 800 counts. However, at 800 counts and above, no correlation between the rmsd of the fit and reported signal intensity counts was found (data not shown). In other cases, the rmsd was particularly high in the initial scans close to the meniscus region, pointing to convection as an origin of the misfit (e.g., S7 Fig). Interestingly, in a few instruments, the interference data exhibited an unexplained pattern of sloping plateaus (e.g., S8 Fig), which required truncation of the radial range used for analysis and correlated with increased errors in the determination of boundary magnitudes (data not shown). Finally, a few interference data sets exhibited wavy patterns superimposed onto the boundary shapes, but without causing excessively high rmsd or outlier s- and M-values (data not shown). Overall, in 11 out of 119 experiments the data acquisition was either not attempted, failed, or produced data sets where the c(s) model did not lead to baseline separation between the BSA monomer and dimer peak; these data sets were not included in the analysis of s-values.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, 20892, United States of America.

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.

No MeSH data available.