Multi‑speed sedimentation velocity implementation in UltraScan‑III

Accepted author manuscriptA 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 implementa- tion 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.Ye

Multi-speed sedimentation velocity simulations with UltraScan-III

Accepted author manuscriptA 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.Ye

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

Recent advances in instrumentation have moved analytical ultracentrifugation (AUC) closer to a possible validation in a Good Manufacturing Practices (GMP) environment. In order for AUC to be validated for a GMP environment, stringent requirements need to be satisfied; analysis procedures must be evaluated for consistency and reproducibility, and GMP capable data acquisition software needs to be developed and validated. These requirements extend to multiple regulatory aspects, covering documentation of instrument hardware functionality, data handling and software for data acquisition and data analysis, process control, audit trails and automation. Here we review the requirements for GMP validation of data acquisition software and illustrate software solutions based on UltraScan that address these requirements as far as they relate to the operation and data handling in conjunction with the latest analytical ultracentrifuge, the Optima AUC by Beckman Coulter. The software targets the needs of regulatory agencies, where AUC plays a critical role in the solution-based characterization of biopolymers and macromolecular assemblies. Biopharmaceutical and regulatory agencies rely heavily on this technique for characterizations of pharmaceutical formulations, biosimilars, injectables, nanoparticles, and other soluble therapeutics. Because of its resolving power, AUC is a favorite application, despite the current lack of GMP validation. We believe that recent advances in standards, hardware, and software presented in this work manage to bridge this gap and allow AUC to be routinely used in a GMP environment. AUC has great potential to provide more detailed information, at higher resolution, and with greater confidence than other analytical techniques, and our software satisfies an urgent need for AUC operation in the GMP environment. The software, including documentation, are publicly available for free download from Github. The multi-platform software is licensed by the LGPL v.3 open source license and supports Windows, Mac and Linux platforms. Installation instructions and a mailing list are available from ultrascan.aucsolutions.com

2D analysis of polydisperse core–shell nanoparticles using analytical ultracentrifugation

Accepted author manuscriptAccurate knowledge of the size, density and composition of nanoparticles (NPs) is of major importance for their applications. In this work the hydrodynamic characterization of polydisperse core–shell NPs by means of analytical ultracentrifugation (AUC) is addressed. AUC is one of the most accurate techniques for the characterization of NPs in the liquid phase because it can resolve particle size distributions (PSDs) with unrivaled resolution and detail. Small NPs have to be considered as core–shell systems when dispersed in a liquid since a solvation layer and a stabilizer shell will significantly contribute to the particle's hydrodynamic diameter and effective density. AUC measures the sedimentation and diffusion transport of the analytes, which are affected by the core–shell compositional properties. This work demonstrates that polydisperse and thus widely distributed NPs pose significant challenges for current state-of-the-art data evaluation methods. The existing methods either have insufficient resolution or do not correctly reproduce the core–shell properties. First, we investigate the performance of different data evaluation models by means of simulated data. Then, we propose a new methodology to address the core–shell properties of NPs. This method is based on the parametrically constrained spectrum analysis and offers complete access to the size and effective density of polydisperse NPs. Our study is complemented using experimental data derived for ZnO and CuInS2 NPs, which do not have a monodisperse PSD. For the first time, the size and effective density of such structures could be resolved with high resolution by means of a two-dimensional AUC analysis approach.Ye

Multi-wavelength analytical ultracentrifugation of human serum albumin complexed with porphyrin

Accepted author manuscriptThe new Beckman Coulter Optima AUC instrument, which features multi-wavelength detection that couples the hydro- dynamic separation of colloidal mixtures to spectral deconvolution of interacting and non-interacting solutes present in a mixture, was used to analyze the composition of human serum albumin (HSA) bound to metallo-protoporphyrin. We present new methods implemented in UltraScan that permit Optima AUC-derived multi-wavelength data to be spectrally decomposed in the same fashion as has been made possible for the Cölfen detector earlier. We demonstrate this approach by spectrally separating sedimentation velocity experimental data from mixtures of apo-HSA and HSA complexed to dif- ferent metallo-protoporphyrins. We further demonstrate how multi-wavelength AUC can accurately recover percentages of metallo-protoporphyrin-bound HSA and apo-HSA from mixtures and how multi-wavelength AUC permits the calculation of molar extinction coefficients for porphyrins bound to HSA. The presented method has broad applicability to other complex systems where mixtures of molecules with different spectral properties need to be characterized.Ye

On Enabling Hydrodynamics Data Analysis of Analytical Ultracentrifugation Experiments

A new modular approach to enable scientific applications with UNICORE has been designed which employs modern elements of seamless web based access for abstraction and encapsulation. The approach uses the Apache Airavata software framework which is applicable and used by a wide variety of scientific gateways, which represent a community-oriented web based interface to computing and storage resources. Scientific gateways have shown a broad impact in computational science and brought many advances to various scientific communities in the last decade and even more research advances are expected when using them with the everincreasing amounts of large quantities of datasets that are often referred to as ’big data’. This paper provides insights into benefits for the UNICORE community in working with existing scientific gateways and their strong user communities. While this new approach is more general in nature, we offer throughout the paper a concrete example of how initial work with scientific gateway communities enable a hydrodynamics data analysis of analytical ultracentrifugation experiments. The approach improves the ease of use in using UNICORE by performing seamlessly computational science so that more scientists can benefit from the strong UNICORE capabilities and its underlying computational resources. Furthermore the approach allows for creating ’collaborative workspaces’ specifically optimized for specific scientific communities, including data and computing resources and to make existing datasets accessible and useful to the broader scientific communities that use scientific gateways on a daily basis. The design approach has been tested in a real supercomputer deploment which has been used by several researchers of a larger scientific community in the bio-chemistry field. We report first results of the inter-working of UNICORE with a specific scientific gateway while not losing sight of the more general applicability of the approach with other gateways

Advancements of the UltraScan scientific gateway for open standards-based cyberinfrastructures

The UltraScan data analysis application is a software package that is able to take advantage of computational resources in order to support the interpretation of analytical ultracentrifugation experiments. Since 2006, the UltraScan scientific gateway has been used with Web browsers in TeraGrid by scientists studying the solution properties of biological and synthetic molecules. UltraScan supports its users with a scientific gateway in order to leverage the power of supercomputing. In this contribution, we will focus on several advancements of the UltraScan scientific gateway architecture with a standardized job management while retaining its lightweight design and end user interaction experience. This paper also presents insights into a production deployment of UltraScan in Europe. The approach is based on open standards with respect to job management and submissions to the Extreme Science and Engineering Discovery Environment in the USA and to similar infrastructures in Europe such as the European Grid Infrastructure or the Partnership for Advanced Computing in Europe (PRACE). Our implementation takes advantage of the Apache Airavata framework for scientific gateways that lays the foundation for easy integration into several other scientific gateways

Improvements of the UltraScan scientific gateway to enable computational jobs on large-scale and open-standards based cyberinfrastructures

The UltraScan data analysis application is a software package that is able to take advantage of computational resources in order to support the interpretation of analytical ultracentrifugation (AUC) experiments. Since 2006, the UltraScan scientific gateway has been used with ordinary Web browsers in TeraGrid by scientists studying the solution properties of biological and synthetic molecules. Unlike other applications, UltraScan is implemented on a gateway architecture and leverages the power of supercomputing to extract very high resolution information from the experimental data. In this contribution, we will focus on several improvements of the UltraScan scientific gateway that enable a standardized job submission and management to computational resources while retaining its lightweight design in order to not disturb the established workflows of its end-users. This paper further presents a walkthrough of the architectural design including one real installation deployment of UltraScan in Europe. The aim is to provide evidence for the added value of open standards and resulting interoperability enabling not only UltraScan application submissions to resources offered in the US cyber infrastructure Extreme Science and Engineering Discovery Environment (XSEDE), but also submissions to similar infrastructures in Europe and around the world. The use of the Apache Airavata framework for scientific gateways within our approach bears the potential to have an impact on several other scientific gateways too

Spectral and Hydrodynamic Analysis of West Nile Virus RNA–Protein Interactions by Multiwavelength Sedimentation Velocity in the Analytical Ultracentrifuge

Interactions between nucleic acids and proteins are critical for many cellular processes, and their study is of utmost importance to many areas of biochemistry, cellular biology, and virology. Here, we introduce a new analytical method based on sedimentation velocity (SV) analytical ultracentrifugation, in combination with a novel multiwavelength detector to characterize such interactions. We identified the stoichiometry and molar mass of a complex formed during the interaction of a West Nile virus RNA stem loop structure with the human T cell-restricted intracellular antigen-1 related protein. SV has long been proven as a powerful technique for studying dynamic assembly processes under physiological conditions in solution. Here, we demonstrate, for the first time, how the new multiwavelength technology can be exploited to study protein–RNA interactions, and show how the spectral information derived from the new detector complements the traditional hydrodynamic information from analytical ultracentrifugation. Our method allows the protein and nucleic acid signals to be separated by spectral decomposition such that sedimentation information from each individual species, including any complexes, can be clearly identified based on their spectral signatures. The method presented here extends to any interacting system where the interaction partners are spectrally separable