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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.


Correlation between the ratio of uncorrected s-values for the BSA monomer peak from the simultaneously acquired interference and the absorbance data (sIF/sABS) and the ratio of corresponding radial magnification correction factors measured independently with the steel mask (rIF/rABS).Squares are a histogram with frequency as indicated in the sidebar. The dotted line indicates the ideally expected relationship assuming perfect measurements of s-values and radial magnification correction factors.
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pone.0126420.g004: Correlation between the ratio of uncorrected s-values for the BSA monomer peak from the simultaneously acquired interference and the absorbance data (sIF/sABS) and the ratio of corresponding radial magnification correction factors measured independently with the steel mask (rIF/rABS).Squares are a histogram with frequency as indicated in the sidebar. The dotted line indicates the ideally expected relationship assuming perfect measurements of s-values and radial magnification correction factors.

Mentions: Radial magnification errors will affect directly and proportionally the measurement of sedimentation velocity, and in those instruments where data from both optical systems are available, the radial magnification errors will be different for the different optical systems. By contrast, the uncorrected s-values from the BSA monomer peak in c(s) analysis, measured either in the absorbance system or in the interference system, are subject to the same errors in scan time, solvent viscosity (temperature), and the same effects of imperfect alignment- or temperature-driven convection. Thus, one would expect that the ratio of the uncorrected s-values, sIF/sABS, to be proportional to the ratio of radial magnification correction factors, rr,IF/r r,ABS. A plot of both quantities, for 42 instruments for which this data is available, is shown in Fig 4. The strong correlation independently validates the veracity of the radial magnification correction factors determined from the analysis of the mask scans.


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)

Correlation between the ratio of uncorrected s-values for the BSA monomer peak from the simultaneously acquired interference and the absorbance data (sIF/sABS) and the ratio of corresponding radial magnification correction factors measured independently with the steel mask (rIF/rABS).Squares are a histogram with frequency as indicated in the sidebar. The dotted line indicates the ideally expected relationship assuming perfect measurements of s-values and radial magnification correction factors.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0126420.g004: Correlation between the ratio of uncorrected s-values for the BSA monomer peak from the simultaneously acquired interference and the absorbance data (sIF/sABS) and the ratio of corresponding radial magnification correction factors measured independently with the steel mask (rIF/rABS).Squares are a histogram with frequency as indicated in the sidebar. The dotted line indicates the ideally expected relationship assuming perfect measurements of s-values and radial magnification correction factors.
Mentions: Radial magnification errors will affect directly and proportionally the measurement of sedimentation velocity, and in those instruments where data from both optical systems are available, the radial magnification errors will be different for the different optical systems. By contrast, the uncorrected s-values from the BSA monomer peak in c(s) analysis, measured either in the absorbance system or in the interference system, are subject to the same errors in scan time, solvent viscosity (temperature), and the same effects of imperfect alignment- or temperature-driven convection. Thus, one would expect that the ratio of the uncorrected s-values, sIF/sABS, to be proportional to the ratio of radial magnification correction factors, rr,IF/r r,ABS. A plot of both quantities, for 42 instruments for which this data is available, is shown in Fig 4. The strong correlation independently validates the veracity of the radial magnification correction factors determined from the analysis of the mask scans.

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.