Multi-speed sedimentation velocity implementation in UltraScan-III

Abstract

A framework for the global analysis of multi-speed analytical ultracentrifugation sedimentation velocity experiments is presented. We discuss extensions to the adaptive space-time finite element fitting methods implemented in UltraScan-III to model sedimentation velocity experiments where a single run is performed at multiple rotor speeds, and describe extensions in the optimization routines used for fitting experimental data collected at arbitrary multi-speed profiles. Our implementation considers factors such as speed dependent rotor stretching, the resulting radial shifting of the finite element solution's boundary conditions, and changes in the associated time-invariant noise. We also address the calculation of acceleration rates and acceleration zones from existing radial acceleration and time records, as well as utilization of the time state object available at high temporal resolution from the new Beckman Optima AUC instrument. Analysis methods in UltraScan-III support unconstrained models that extract reliable information for both the sedimentation and the diffusion coefficients. These methods do not rely on any assumptions and allow for arbitrary variations in both sedimentation and diffusion transport. We have adapted these routines for the multi-speed case, and developed optimized and general grid based fitting methods to handle changes in the information content of the simulation matrix for different speed steps. New graphical simulation tools are presented that assist the investigator to estimate suitable grid metrics and evaluate information content based on edit profiles for individual experiments.

Moving analytical ultracentrifugation software to a good manufacturing practices (GMP) environment

“…Unlike the older Beckman Proteomelab XLI/XLA data acquisition software, which only recorded scan times and ω 2 t integrals for each collected scan, UltraScan provides a scan-independent timestate object, which is part of the openAUC data format [16] and is further discussed in [17]. In the Optima, it provides a second-by-second accounting of set and actual rotor speed, speed step, time, ω 2 t integrals, temperature and scan frequency, starting with the first second of acceleration.…”

Practical Aspects of Multiwavelength Analytical Ultracentrifugation

“…For this class of analytes, rotor speeds are typically set between 30 and 50 krpm and the number of radial point acquisitions between 15 to 20, while still limiting scan times to between 1 and 2 min. A recent publication explored the relationship of analyte hydrodynamics, AUC rotor speed and data fitting through modeling of simulation data, suggesting a path to optimal rotor speed settings that minimize uncertainty of both s and D parameters [29]. Alternatively, one may choose to run the experiment twice, once at high speed, where the sedimentation is maximized and the best resolution of species in solution is enabled, allowing for optimal determination of the sedimentation coefficient.…”

“…A subsequent experiment at lower speed can then be used to estimate the diffusion information. Having determined the number of solutes and corresponding s-values, these can be fixed in the analysis, and the fitting floated for D. Other researchers have implemented multispeed methods, where the rotor speed is increased at intervals within the experiment [20,29]. These methods have the same goal of optimizing both s and D parameters, but aim to do so within a single experiment.…”

Generalized Subband Analysis and Signal Synthesis

“…when subbands are adjacent to each other and cover the entire domain. Obviously, this corresponds to obtaining components of the vector that will be analyzed, which meet th e requirement of components additivity (4). The problem of signal synthesis with specified properties is often considered during transmission of information and remote control.…”