A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

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.

3D-Printing for Analytical Ultracentrifugation

“…Related, surface adsorption can be a problem when studying protein interactions in the sub-nanomolar range with fluorescence detection [30], and further studies will be required to gain experience with beneficial or detrimental properties of different materials in this and other applications of AUC. Perhaps the most surprising result of the present work was the successful use of 3D printed centerpieces in high-quality SV AUC experiments; with results for the BSA monomer virtually identical to those carried out in commercial epoxy centerpieces and within the limits of the large multi-laboratory benchmark study recently published [51]. We did observe significantly higher standard deviation of the BSA monomer sedimentation coefficient, but variation in replicate experiments was still less than 1%.…”

“…AUC experiments were carried out in an Optima XL-A (Beckman Coulter, Indianapolis, IN), calibrated as previously described [50,51]. Data were acquired using the installed absorbance detection, or, alternatively, a confocal fluorescence detection system (FDS, Aviv Biomedical, Lakewood NJ) equipped with either a 10 mW solid state laser exciting at 488 nm and emission bandpass filter from 505 nm to 565 nm, or an adjustable diode laser exciting at 561 nm and a long-pass emission filter at 580 nm.…”

Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism

“…This s 20,w change is similar to the difference seen for the s 20,w value for C3b of 7.40 S in 137 mM NaCl, which increased to 7.60 S in 50 mM NaCl [10]. The difference in s 20,w values between 50 and 137 mM NaCl is greater than the expected precision of these measurements of ±0.1 S determined from measurements on 79 different AUC instruments [41], although it is noted that all measurements here were made on the same AUC instrument. The molecular masses from the c(s) peaks corresponded to 185 kDa, which agreed well with the expected value.…”

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

“…[isodesmic] with A being the TDP‐43 NTD monomer, and K D iso the single dissociation constant for the association steps were obtained from the analysis. Errors of the fits represent the 68.3% confidence interval (CI) using an automated surface projection method (Zhao et al , ). All plots were generated with the program GUSSI (kindly provided by Dr. Chad Brautigam).…”