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.


Corrected best-fit apparent monomer molecular mass from integration of the c(s) peak when scanned with the absorbance system (green) and the interference system (magenta).Only data with rmsd less than 0.01 OD or 0.01 fringes were included. 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.g013: Corrected best-fit apparent monomer molecular mass from integration of the c(s) peak when scanned with the absorbance system (green) and the interference system (magenta).Only data with rmsd less than 0.01 OD or 0.01 fringes were included. 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: Finally, it is of interest to study how reproducibly the parameters characterizing the boundary broadening can be determined. For this, we only considered fits that led to an rmsd value of less than 0.01 OD or 0.01 fringes, respectively. The apparent molar mass measured by the absorbance and interference system was (60.6 ± 3.2) kg/mol and (70.5 ± 8.1) kg/mol, respectively. Despite the close correspondence of the s20T,t,r,v-values with (4.328 ± 0.025) S and (4.317 ± 0.035) S for the absorbance and interference system, respectively, surprisingly, there is a clear difference between their apparent molar mass values (Fig 13). It originates from shallower measured boundaries in the absorbance system as compared to the interference system, which resulted in uncorrected best-fit frictional ratios of 1.29 ± 0.05 and 1.44 ± 0.13, respectively. The magnitude of this difference in broadening is far greater than the calculated impact of finite scan speed in the absorbance system on apparent boundary broadening, which for the current scan settings and s-value would be negligible [36]. The origin of the difference is unclear, although one could speculate that it may be related to differences in the optical adjustment, such as the focal point, leading to differences in the radial resolution of the two optical systems, and/or non-linear signal distortions of the boundary from stray light [19].


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)

Corrected best-fit apparent monomer molecular mass from integration of the c(s) peak when scanned with the absorbance system (green) and the interference system (magenta).Only data with rmsd less than 0.01 OD or 0.01 fringes were included. 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.g013: Corrected best-fit apparent monomer molecular mass from integration of the c(s) peak when scanned with the absorbance system (green) and the interference system (magenta).Only data with rmsd less than 0.01 OD or 0.01 fringes were included. 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: Finally, it is of interest to study how reproducibly the parameters characterizing the boundary broadening can be determined. For this, we only considered fits that led to an rmsd value of less than 0.01 OD or 0.01 fringes, respectively. The apparent molar mass measured by the absorbance and interference system was (60.6 ± 3.2) kg/mol and (70.5 ± 8.1) kg/mol, respectively. Despite the close correspondence of the s20T,t,r,v-values with (4.328 ± 0.025) S and (4.317 ± 0.035) S for the absorbance and interference system, respectively, surprisingly, there is a clear difference between their apparent molar mass values (Fig 13). It originates from shallower measured boundaries in the absorbance system as compared to the interference system, which resulted in uncorrected best-fit frictional ratios of 1.29 ± 0.05 and 1.44 ± 0.13, respectively. The magnitude of this difference in broadening is far greater than the calculated impact of finite scan speed in the absorbance system on apparent boundary broadening, which for the current scan settings and s-value would be negligible [36]. The origin of the difference is unclear, although one could speculate that it may be related to differences in the optical adjustment, such as the focal point, leading to differences in the radial resolution of the two optical systems, and/or non-linear signal distortions of the boundary from stray light [19].

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.