The Experts below are selected from a list of 9603 Experts worldwide ranked by ideXlab platform

Peter Schuck - One of the best experts on this subject based on the ideXlab platform.

  • Enhanced Sample Handling for Analytical Ultracentrifugation with 3D-Printed Centerpieces
    2019
    Co-Authors: Chad A Brautigam, Huaying Zhao, Sumit K. Chaturvedi, Mary T. Bollard, Jonathan Krynitsky, John W. Kakareka, Thomas J. Pohida, Peter Schuck
    Abstract:

    The centerpiece of the sample cell assembly in Analytical Ultracentrifugation holds the sample solution between windows, sealed against high vacuum, and is shaped such that macromolecular migration in centrifugal fields exceeding 200 000g can proceed undisturbed by walls or convection while concentration profiles are imaged with optical detection systems aligned perpendicular to the plane of rotation. We have recently shown that 3D printing using various materials allows inexpensive and rapid manufacturing of centerpieces. In the present work, we expand this endeavor to examine the accuracy of the measured sedimentation process, as well as short-term durability of the centerpieces. We find that 3D-printed centerpieces can be used many times and can provide data equivalent in quality to commonly used commercial epoxy resin centerpieces. Furthermore, 3D printing enables novel designs adapted to particular experimental objectives because they offer unique opportunities, for example, to create well-defined curved surfaces, narrow channels, and embossed features. We present examples of centerpiece designs exploiting these capabilities for improved AUC experiments. This includes narrow sector centerpieces that substantially reduce the required sample volume while maintaining the standard optical path length; thin centerpieces with integrated window holders to provide very short optical pathlengths that reduce optical aberrations at high macromolecular concentrations; long-column centerpieces that increase the observable distance of macromolecular migration for higher-precision sedimentation coefficients; and three-sector centerpieces that allow doubling the number of samples in a single run while reducing the sample volumes. We find each of these designs allows unimpeded macromolecular sedimentation and can provide high-quality sedimentation data

  • a radial calibration window for Analytical Ultracentrifugation
    PLOS ONE, 2018
    Co-Authors: Thomas W Lebrun, Jeffrey A Fagan, Peter Schuck, Justine S Yoon, Xianghui Dong, Nicole Y Morgan, Huaying Zhao
    Abstract:

    Analytical Ultracentrifugation (AUC) is a first-principles based method for studying macromolecules and particles in solution by monitoring the evolution of their radial concentration distribution as a function of time in the presence of a high centrifugal field. In sedimentation velocity experiments, hydrodynamic properties relating to size, shape, density, and solvation of particles can be measured, at a high hydrodynamic resolution, on polydisperse samples. In a recent multilaboratory benchmark study including data from commercial Analytical ultracentrifuges in 67 laboratories, the calibration accuracy of the radial dimension was found to be one of the dominant factors limiting the accuracy of AUC. In the present work, we develop an artifact consisting of an accurately calibrated reflective pattern lithographically deposited onto an AUC window. It serves as a reticle when scanned in AUC control experiments for absolute calibration of radial magnification. After analysis of the pitch between landmarks in scans using different optical systems, we estimate that the residual uncertainty in radial magnification after external calibration with the radial scale artifact is ≈0.2 %, of similar magnitude to other important contributions after external calibration such as the uncertainty in temperature and time. The previous multilaboratory study had found many instruments with errors in radial measurements of 1 % to 2 %, and a few instruments with errors in excess of 15 %, meaning that the use of the artifact developed here could reduce errors by 5-to 10-fold or more. Adoption of external radial calibration is thus an important factor for assuring accuracy in studies related to molecular hydrodynamics and particle size measurements by AUC.

  • identification of nanomaterials a validation report of two laboratories using Analytical Ultracentrifugation with fixed and ramped speed options
    NanoImpact, 2018
    Co-Authors: Dora Mehn, Peter Schuck, Iria M Rioechevarria, Douglas Gilliland, Michael Kaiser, Klaus Vilsmeier, Wendel Wohlleben
    Abstract:

    Abstract The identification of nanomaterials for regulatory purposes in Europe requires a statistically significant comparison of the number metrics median diameter to the 100 nm criterion. Methods should be robust against minor imperfections of the implementation, transferable and reproducible between laboratories, and applicable to a wide range of diameters and material compositions. Sample preparation and metrics conversion are additional challenges that must be included in validation. Here we report on the validation of a protocol for Analytical Ultracentrifugation with fixed and ramped speed, specifically intended to serve the European Commission recommended definition, as relevant for REACH regulation, cosmetics and food labelling and national inventories. All the determined measurement uncertainties remain below 12% for volume and number metrics median diameters, in both laboratories. Traces of sub-100-nm particles with around 2% mass contribution (but > 50% number contribution) were reproducibly quantified. For powders, sample preparation is a critical step, and sonication intensities above 0.4 W/mL are recommended. The ramp operation can reduce user bias by eliminating the choice of options in data acquisition. The identification of nanomaterials and non-nano-materials by AUC in either fixed or ramp speed is consistent with TEM, excluding the platelet Kaolin material, but including the monodisperse silica, multimodal silica, a nanoform and a non-nano-form of BaSO 4 , irregularly shaped CaCO 3 and coated non-nano TiO 2 .

  • fluorescence detected sedimentation velocity Analytical Ultracentrifugation for investigating affinity and stoichiometry of protein interactions
    Biophysical Journal, 2017
    Co-Authors: Huaying Zhao, Sumit Kumar Chaturvedi, Peter Schuck
    Abstract:

    Sedimentation velocity Analytical Ultracentrifugation (SV-AUC) is monitoring the migration of particles in free solution with real-time optical detection. It provides powerful methods for studying multi-component systems of biomacromolecules such as protein and DNA. It offers exquisite resolution regarding the size and shape of the components and population of co-existing complexes, and therefore binding energies and stoichiometries for reversible interacting systems can be resolved. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying molecules at low concentration because of the high sensitivity and selectivity of fluorescence. In order to make FDS-SV a robust and quantitative method for studying macromolecules, we have previously developed computational tools to specifically account for the unique data structure posed by the confocal design and other geometric factors. Such developments allow FDS-SV to be applied to high-affinity binding systems at concentrations well into the picomolar regime. Recently we extended FDS-SV to multi-dimensional signal detection by employing photoswitchable fluorescent proteins and taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching. Thus, FDS-SV is an excellent tool for the study of high-affinity protein interactions. We will show applications to protein-protein and protein-nucleic acid interactions with multiple states, quantitatively determining the binding affinity, stoichiometry and cooperativity.

  • a new dimension of detection in Analytical Ultracentrifugation with fluorescence detection using photoswitchable fps as time domain probes
    Biophysical Journal, 2016
    Co-Authors: Huaying Zhao, George H Patterson, Peter Schuck
    Abstract:

    Multi-component protein complex formation is of great interest in physiological and biochemical studies as it is ubiquitous in biological systems. Analytical Ultracentrifugation (AUC) provides powerful methods for studying such systems. It offers information on size, shape and binding energies for interacting systems from the analysis of sedimentation profiles of molecular mixtures in free solution. By virtue of the superb hydrodynamic resolution achieved in sedimentation velocity, co-existing complexes can be identified, even in the presence of impurities and aggregates. A recently introduced fluorescence optical detection system (FDS) for AUC substantially extends capabilities for studying high-affinity protein interactions because of the selectivity of fluorescence high sensitivity, allowing studies at picomolar concentrations. To overcome limitations posed by a single excitation wavelength, in the current study, we employed photoswitchable fluorescent proteins (FP) as probes in FDS-AUC. Taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching during FDS-AUC, a new dimension of detection is created that allows resolving specific FPs in multi-component mixtures. We have developed a computational framework for the analysis of sedimentation data exploiting the new temporal dimension. Experimentally we demonstrated this approach using mixtures of FPs commonly used in imaging, which could be readily distinguished from experimental FDS data. This approach extends FDS-AUC to more complicated protein system with multiple components and allows for quantitative characterization of protein complexes with regard to the binding mechanism and stoichiometry under very low macromolecular concentrations.

Borries Demeler - One of the best experts on this subject based on the ideXlab platform.

  • moving Analytical Ultracentrifugation software to a good manufacturing practices gmp environment
    PLOS Computational Biology, 2020
    Co-Authors: Alexey Savelyev, Borries Demeler, Gary E Gorbet, Amy Henrickson
    Abstract:

    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.

  • Analytical Ultracentrifugation data analysis with ultrascan iii
    2016
    Co-Authors: Borries Demeler, Gary E Gorbet
    Abstract:

    The current status of the UltraScan-III (US3) data analysis software suite is described. An overview of the US3 concepts, software layout, the data workflows and US3 components is presented, followed by a discussion of the analysis methods and their applications. Also described are visualization modules for analysis results, US3’s utilities and simulation tools, as well as the collaboration environments for online data and result exchange.

  • methods for the design and analysis of sedimentation velocity and sedimentation equilibrium experiments with proteins
    Current protocols in protein science, 2010
    Co-Authors: Borries Demeler
    Abstract:

    Analytical Ultracentrifugation experiments play an integral role in the solution phase characterization of recombinant proteins and other biological macromolecules. This unit discusses the design of sedimentation velocity and sedimentation equilibrium experiments performed with a Beckman Optima XL-A or XL-I Analytical ultracentrifuge. Optimal instrument settings and experimental design considerations are explained, and strategies for the analysis of experimental data with the UltraScan data analysis software package are presented. Special attention is paid to the strengths and weaknesses of the available detectors, and guidance is provided on how to extract maximum information from Analytical Ultracentrifugation experiments.

  • the implementation of somo solution modeller in the ultrascan Analytical Ultracentrifugation data analysis suite enhanced capabilities allow the reliable hydrodynamic modeling of virtually any kind of biomacromolecule
    European Biophysics Journal, 2010
    Co-Authors: Emre H Brookes, Borries Demeler, Camillo Rosano, Mattia Rocco
    Abstract:

    The interpretation of solution hydrodynamic data in terms of macromolecular structural parameters is not a straightforward task. Over the years, several approaches have been developed to cope with this problem, the most widely used being bead modeling in various flavors. We report here the implementation of the SOMO (SOlution MOdeller; Rai et al. in Structure 13:723–734, 2005) bead modeling suite within one of the most widely used Analytical Ultracentrifugation data analysis software packages, UltraScan (Demeler in Modern Analytical Ultracentrifugation: techniques and methods, Royal Society of Chemistry, UK, 2005). The US-SOMO version is now under complete graphical interface control, and has been freed from several constraints present in the original implementation. In the direct beads-per-atoms method, virtually any kind of residue as defined in the Protein Data Bank (e.g., proteins, nucleic acids, carbohydrates, prosthetic groups, detergents, etc.) can be now represented with beads whose number, size and position are all defined in user-editable tables. For large structures, a cubic grid method based on the original AtoB program (Byron in Biophys J 72:408–415, 1997) can be applied either directly on the atomic structure, or on a previously generated bead model. The hydrodynamic parameters are then computed in the rigid-body approximation. An extensive set of tests was conducted to further validate the method, and the results are presented here. Owing to its accuracy, speed, and versatility, US-SOMO should allow to fully take advantage of the potential of solution hydrodynamics as a complement to higher resolution techniques in biomacromolecular modeling.

  • parsimonious regularization using genetic algorithms applied to the analysis of Analytical Ultracentrifugation experiments
    Genetic and Evolutionary Computation Conference, 2007
    Co-Authors: Emre H Brookes, Borries Demeler
    Abstract:

    Frequently in the physical sciences experimental data are analyzed to determine model parameters using techniques known as parameter estimation. Eliminating the effects of noise from experimental data often involves Tikhonov or Maximum-Entropy regularization. These methods introduce a bias which smoothes the solution. In the problems considered here, the exact answer is sharp, containing a sparse set of parameters. Therefore, it is desirable to find the simplest set of model parameters for the data with an equivalent goodness-of-fit. This paper explains how to bias the solution towards a parsimonious model with a careful application of Genetic Algorithms. A method of representation, initialization and mutation is introduced to efficiently find this model. The results are compared with results from two other methods on simulated data with known content. Our method is shown to be the only one to achieve the desired results. Analysis of Analytical Ultracentrifugation sedimentation velocity experimental data is the primary example application.

Huaying Zhao - One of the best experts on this subject based on the ideXlab platform.

  • Enhanced Sample Handling for Analytical Ultracentrifugation with 3D-Printed Centerpieces
    2019
    Co-Authors: Chad A Brautigam, Huaying Zhao, Sumit K. Chaturvedi, Mary T. Bollard, Jonathan Krynitsky, John W. Kakareka, Thomas J. Pohida, Peter Schuck
    Abstract:

    The centerpiece of the sample cell assembly in Analytical Ultracentrifugation holds the sample solution between windows, sealed against high vacuum, and is shaped such that macromolecular migration in centrifugal fields exceeding 200 000g can proceed undisturbed by walls or convection while concentration profiles are imaged with optical detection systems aligned perpendicular to the plane of rotation. We have recently shown that 3D printing using various materials allows inexpensive and rapid manufacturing of centerpieces. In the present work, we expand this endeavor to examine the accuracy of the measured sedimentation process, as well as short-term durability of the centerpieces. We find that 3D-printed centerpieces can be used many times and can provide data equivalent in quality to commonly used commercial epoxy resin centerpieces. Furthermore, 3D printing enables novel designs adapted to particular experimental objectives because they offer unique opportunities, for example, to create well-defined curved surfaces, narrow channels, and embossed features. We present examples of centerpiece designs exploiting these capabilities for improved AUC experiments. This includes narrow sector centerpieces that substantially reduce the required sample volume while maintaining the standard optical path length; thin centerpieces with integrated window holders to provide very short optical pathlengths that reduce optical aberrations at high macromolecular concentrations; long-column centerpieces that increase the observable distance of macromolecular migration for higher-precision sedimentation coefficients; and three-sector centerpieces that allow doubling the number of samples in a single run while reducing the sample volumes. We find each of these designs allows unimpeded macromolecular sedimentation and can provide high-quality sedimentation data

  • a radial calibration window for Analytical Ultracentrifugation
    PLOS ONE, 2018
    Co-Authors: Thomas W Lebrun, Jeffrey A Fagan, Peter Schuck, Justine S Yoon, Xianghui Dong, Nicole Y Morgan, Huaying Zhao
    Abstract:

    Analytical Ultracentrifugation (AUC) is a first-principles based method for studying macromolecules and particles in solution by monitoring the evolution of their radial concentration distribution as a function of time in the presence of a high centrifugal field. In sedimentation velocity experiments, hydrodynamic properties relating to size, shape, density, and solvation of particles can be measured, at a high hydrodynamic resolution, on polydisperse samples. In a recent multilaboratory benchmark study including data from commercial Analytical ultracentrifuges in 67 laboratories, the calibration accuracy of the radial dimension was found to be one of the dominant factors limiting the accuracy of AUC. In the present work, we develop an artifact consisting of an accurately calibrated reflective pattern lithographically deposited onto an AUC window. It serves as a reticle when scanned in AUC control experiments for absolute calibration of radial magnification. After analysis of the pitch between landmarks in scans using different optical systems, we estimate that the residual uncertainty in radial magnification after external calibration with the radial scale artifact is ≈0.2 %, of similar magnitude to other important contributions after external calibration such as the uncertainty in temperature and time. The previous multilaboratory study had found many instruments with errors in radial measurements of 1 % to 2 %, and a few instruments with errors in excess of 15 %, meaning that the use of the artifact developed here could reduce errors by 5-to 10-fold or more. Adoption of external radial calibration is thus an important factor for assuring accuracy in studies related to molecular hydrodynamics and particle size measurements by AUC.

  • fluorescence detected sedimentation velocity Analytical Ultracentrifugation for investigating affinity and stoichiometry of protein interactions
    Biophysical Journal, 2017
    Co-Authors: Huaying Zhao, Sumit Kumar Chaturvedi, Peter Schuck
    Abstract:

    Sedimentation velocity Analytical Ultracentrifugation (SV-AUC) is monitoring the migration of particles in free solution with real-time optical detection. It provides powerful methods for studying multi-component systems of biomacromolecules such as protein and DNA. It offers exquisite resolution regarding the size and shape of the components and population of co-existing complexes, and therefore binding energies and stoichiometries for reversible interacting systems can be resolved. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying molecules at low concentration because of the high sensitivity and selectivity of fluorescence. In order to make FDS-SV a robust and quantitative method for studying macromolecules, we have previously developed computational tools to specifically account for the unique data structure posed by the confocal design and other geometric factors. Such developments allow FDS-SV to be applied to high-affinity binding systems at concentrations well into the picomolar regime. Recently we extended FDS-SV to multi-dimensional signal detection by employing photoswitchable fluorescent proteins and taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching. Thus, FDS-SV is an excellent tool for the study of high-affinity protein interactions. We will show applications to protein-protein and protein-nucleic acid interactions with multiple states, quantitatively determining the binding affinity, stoichiometry and cooperativity.

  • a new dimension of detection in Analytical Ultracentrifugation with fluorescence detection using photoswitchable fps as time domain probes
    Biophysical Journal, 2016
    Co-Authors: Huaying Zhao, George H Patterson, Peter Schuck
    Abstract:

    Multi-component protein complex formation is of great interest in physiological and biochemical studies as it is ubiquitous in biological systems. Analytical Ultracentrifugation (AUC) provides powerful methods for studying such systems. It offers information on size, shape and binding energies for interacting systems from the analysis of sedimentation profiles of molecular mixtures in free solution. By virtue of the superb hydrodynamic resolution achieved in sedimentation velocity, co-existing complexes can be identified, even in the presence of impurities and aggregates. A recently introduced fluorescence optical detection system (FDS) for AUC substantially extends capabilities for studying high-affinity protein interactions because of the selectivity of fluorescence high sensitivity, allowing studies at picomolar concentrations. To overcome limitations posed by a single excitation wavelength, in the current study, we employed photoswitchable fluorescent proteins (FP) as probes in FDS-AUC. Taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching during FDS-AUC, a new dimension of detection is created that allows resolving specific FPs in multi-component mixtures. We have developed a computational framework for the analysis of sedimentation data exploiting the new temporal dimension. Experimentally we demonstrated this approach using mixtures of FPs commonly used in imaging, which could be readily distinguished from experimental FDS data. This approach extends FDS-AUC to more complicated protein system with multiple components and allows for quantitative characterization of protein complexes with regard to the binding mechanism and stoichiometry under very low macromolecular concentrations.

  • variable field Analytical Ultracentrifugation ii gravitational sweep sedimentation velocity
    Biophysical Journal, 2016
    Co-Authors: Huaying Zhao, Julia Sandmaier, Alexander J Liddle, Peter Schuck
    Abstract:

    Sedimentation velocity (SV) Analytical Ultracentrifugation is a classical biophysical technique for the determination of the size-distribution of macromolecules, macromolecular complexes, and nanoparticles. SV has traditionally been carried out at a constant rotor speed, which limits the range of sedimentation coefficients that can be detected in a single experiment. Recently we have introduced methods to implement experiments with variable rotor speeds, in combination with variable field solutions to the Lamm equation, with the application to expedite the approach to sedimentation equilibrium. Here, we describe the use of variable-field sedimentation analysis to increase the size-range covered in SV experiments by ∼100-fold with a quasi-continuous increase of rotor speed during the experiment. Such a gravitational-sweep sedimentation approach has previously been shown to be very effective in the study of nanoparticles with large size ranges. In the past, diffusion processes were not accounted for, thereby posing a lower limit of particle sizes and limiting the accuracy of the size distribution. In this work, we combine variable field solutions to the Lamm equation with diffusion-deconvoluted sedimentation coefficient distributions c(s), which further extend the macromolecular size range that can be observed in a single SV experiment while maintaining accuracy and resolution. In this way, approximately five orders of magnitude of sedimentation coefficients, or eight orders of magnitude of particle mass, can be probed in a single experiment. This can be useful, for example, in the study of proteins forming large assemblies, as in fibrillation process or capsid self-assembly, in studies of the interaction between very dissimilar-sized macromolecular species, or in the study of broadly distributed nanoparticles.

Gary G Adams - One of the best experts on this subject based on the ideXlab platform.

  • Analytical Ultracentrifugation in saliva research: Impact of green tea astringency and its significance on the in-vivo aroma release
    Scientific Reports, 2018
    Co-Authors: Vlad Dinu, Chujiao Liu, Joseph Ali, Charfedinne Ayed, Gary G Adams, Pavel Gershkovich, Stephen E Harding, Ian D. Fisk
    Abstract:

    Current saliva testing methods rely on cutting edge yet expensive techniques for the detection and analysis of genetic material, proteins and biomarkers for clinical use. However, these techniques are limited in scope and often cannot be used with complex food materials. We propose an efficient ex-vivo tool for evaluating biologically relevant interactions between food components and human saliva using sedimentation velocity Analytical Ultracentrifugation (SV-AUC). We evaluated macromolecular content from “unstimulated” (US) and “stimulated” (SS) samples pooled from 5 healthy volunteers. Over 90% of total saliva protein consisted of α-amylase and mucin, and up to 10% was secretory immunoglobulin A (SIgA). It was shown that α-amylase concentration increased upon parafilm stimulation, which lead to a decrease in the viscosity of saliva. Then, we used a simple food system (green tea) to evaluate changes in the salivary protein content caused by green tea polyphenols. It was found that aroma release from green tea is highly influenced by interactions between α-amylase and polyphenol epigallocatechin 3-gallate (EGCG). This interaction was found to increase the viscosity of the salivary bulk, suggested to contribute to astringency, and increased the concentrations of β-ionone, benzaldehyde and isovaleraldehyde (P 

  • Analytical Ultracentrifugation in saliva research impact of green tea astringency and its significance on the in vivo aroma release
    Scientific Reports, 2018
    Co-Authors: Vlad Dinu, Charfedinne Ayed, Gary G Adams, Pavel Gershkovich, Stephen E Harding, Ian D. Fisk
    Abstract:

    Current saliva testing methods rely on cutting edge yet expensive techniques for the detection and analysis of genetic material, proteins and biomarkers for clinical use. However, these techniques are limited in scope and often cannot be used with complex food materials. We propose an efficient ex-vivo tool for evaluating biologically relevant interactions between food components and human saliva using sedimentation velocity Analytical Ultracentrifugation (SV-AUC). We evaluated macromolecular content from “unstimulated” (US) and “stimulated” (SS) samples pooled from 5 healthy volunteers. Over 90% of total saliva protein consisted of α-amylase and mucin, and up to 10% was secretory immunoglobulin A (SIgA). It was shown that α-amylase concentration increased upon parafilm stimulation, which lead to a decrease in the viscosity of saliva. Then, we used a simple food system (green tea) to evaluate changes in the salivary protein content caused by green tea polyphenols. It was found that aroma release from green tea is highly influenced by interactions between α-amylase and polyphenol epigallocatechin 3-gallate (EGCG). This interaction was found to increase the viscosity of the salivary bulk, suggested to contribute to astringency, and increased the concentrations of β-ionone, benzaldehyde and isovaleraldehyde (P < 0.01), suggested to play a significant role in the characteristic flavour of green tea.

  • assessing sedimentation equilibrium profiles in Analytical Ultracentrifugation experiments on macromolecules from simple average molecular weight analysis to molecular weight distribution and interaction analysis
    Biophysical Reviews, 2016
    Co-Authors: Stephen E Harding, Gary G Adams, Richard B Gillis
    Abstract:

    Molecular weights (molar masses), molecular weight distributions, dissociation constants and other interaction parameters are fundamental characteristics of proteins, nucleic acids, polysaccharides and glycoconjugates in solution. Sedimentation equilibrium Analytical Ultracentrifugation provides a powerful method with no supplementary immobilization, columns or membranes required. It is a particularly powerful tool when used in conjunction with its sister technique, namely sedimentation velocity. Here, we describe key approaches now available and their application to the characterization of antibodies, polysaccharides and glycoconjugates. We indicate how major complications, such as thermodynamic non-ideality, can now be routinely dealt with, thanks to a great extent to the extensive contribution of Professor Don Winzor over several decades of research.

Andrea Balbo - One of the best experts on this subject based on the ideXlab platform.

  • measurement of the temperature of the resting rotor in Analytical Ultracentrifugation
    Analytical Biochemistry, 2014
    Co-Authors: Rodolfo Ghirlando, Andrea Balbo, Huaying Zhao, Grzegorz Piszczek, Ute Curth, Chad A Brautigam, Peter Schuck
    Abstract:

    Accurate measurements of rotor temperature are critical for the interpretation of hydrodynamic parameters in Analytical Ultracentrifugation. We have recently developed methods for a more accurate determination of the temperature of a spinning rotor using iButton temperature loggers. Here we report that the temperature measured with the iButton on the counterbalance of a resting rotor, following thermal equilibration under high vacuum, closely corresponded to the temperature of the spinning rotor with a precision better than 0.2°C. This strategy offers an inexpensive and straightforward approach to monitor the accuracy of the temperature calibration and determine corrective temperature offsets.

  • a bayesian approach for quantifying trace amounts of antibody aggregates by sedimentation velocity Analytical Ultracentrifugation
    Aaps Journal, 2008
    Co-Authors: P Brown, Andrea Balbo, Peter Schuck
    Abstract:

    Sedimentation velocity Analytical Ultracentrifugation (SV-AUC) has become an important tool for the characterization of the purity of protein therapeutics. The work presented here addresses a need for methods orthogonal to size-exclusion chromatography for ensuring the reliable quantitation of immunogenic oligomers, for example, in antibody preparations. Currently the most commonly used approach for SV-AUC analysis is the diffusion-deconvoluted sedimentation coefficient distribution c(s) method, previously developed by us as a general purpose technique and implemented in the software SEDFIT. In both practical and theoretical studies, different groups have reported a sensitivity of c(s) for trace oligomeric fractions well below the 1% level. In the present work we present a variant of c(s) designed for the purpose of trace detection, with customized Bayesian regularization. The original c(s) method relies on maximum entropy regularization providing the most parsimonious distribution consistent with the data. In the present paper, we use computer simulations of an antibody system as example to demonstrate that the standard maximum entropy regularization, due to its design, leads to a theoretical lower limit for the detection of oligomeric traces and a consistent underestimate of the trace populations by ∼0.1% (dependent on the level of regularization). This can be overcome with a recently developed Bayesian extension of c(s) (Brown et al., Biomacromolecules, 8:2011–2024, 2007), utilizing the known regions of sedimentation coefficients for the monomer and oligomers of interest as prior expectation for the peak positions in the distribution. We show that this leads to more clearly identifiable and consistent peaks and lower theoretical limits of quantization by approximately an order of magnitude for some experimental conditions. Implications for the experimental design of SV-AUC and practical detection limits are discussed.

  • characterizing protein protein interactions by sedimentation velocity Analytical Ultracentrifugation
    Current protocols in immunology, 2008
    Co-Authors: P Brown, Andrea Balbo, Peter Schuck
    Abstract:

    This unit introduces the basic principles and practice of sedimentation velocity Analytical Ultracentrifugation for the study of reversible protein interactions, such as the characterization of self-association, heterogeneous association, multi-protein complexes, binding stoichiometry, and the determination of association constants. The Analytical tools described include sedimentation coefficient and molar mass distributions, multi-signal sedimentation coefficient distributions, Gilbert-Jenkins theory, different forms of isotherms, and global Lamm equation modeling. Concepts for the experimental design are discussed, and a detailed step-by-step protocol guiding the reader through the experiment and the data analysis is available as an Internet resource. Curr. Protoc. Immunol. 81:18.15.1-18.15.39. © 2008 by John Wiley & Sons, Inc. Keywords: sedimentation equilibrium; sedimentation velocity; chemical equilibria; reversible interactions; multi-protein complex; Analytical Ultracentrifugation; size-distribution; Gilbert-Jenkins theory; Lamm equation; Bayesian analysis

  • characterizing protein protein interactions by sedimentation velocity Analytical Ultracentrifugation
    Current protocols in immunology, 2008
    Co-Authors: Patrick O Brown, Andrea Balbo, Peter Schuck
    Abstract:

    This unit introduces the basic principles and practice of sedimentation velocity Analytical Ultracentrifugation for the study of reversible protein interactions, such as the characterization of self-association, heterogeneous association, multi-protein complexes, binding stoichiometry, and the determination of association constants. The Analytical tools described include sedimentation coefficient and molar mass distributions, multi-signal sedimentation coefficient distributions, Gilbert-Jenkins theory, different forms of isotherms, and global Lamm equation modeling. Concepts for the experimental design are discussed, and a detailed step-by-step protocol guiding the reader through the experiment and the data analysis is available as an Internet resource.

  • using prior knowledge in the determination of macromolecular size distributions by Analytical Ultracentrifugation
    Biomacromolecules, 2007
    Co-Authors: P Brown, Andrea Balbo, Peter Schuck
    Abstract:

    Analytical Ultracentrifugation has reemerged as a widely used tool for the study of ensembles of biological macromolecules to understand, for example, their size-distribution and interactions in free solution. Such information can be obtained from the mathematical analysis of the concentration and signal gradients across the solution column and their evolution in time generated as a result of the gravitational force. In sedimentation velocity Analytical Ultracentrifugation, this analysis is frequently conducted using high resolution, diffusion-deconvoluted sedimentation coefficient distributions. They are based on Fredholm integral equations, which are ill-posed unless stabilized by regularization. In many fields, maximum entropy and Tikhonov-Phillips regularization are well-established and powerful approaches that calculate the most parsimonious distribution consistent with the data and prior knowledge, in accordance with Occam's razor. In the implementations available in Analytical Ultracentrifugation, to date, the basic assumption implied is that all sedimentation coefficients are equally likely and that the information retrieved should be condensed to the least amount possible. Frequently, however, more detailed distributions would be warranted by specific detailed prior knowledge on the macromolecular ensemble under study, such as the expectation of the sample to be monodisperse or paucidisperse or the expectation for the migration to establish a bimodal sedimentation pattern based on Gilbert-Jenkins' theory for the migration of chemically reacting systems. So far, such prior knowledge has remained largely unused in the calculation of the sedimentation coefficient or molecular weight distributions or was only applied as constraints. In the present paper, we examine how prior expectations can be built directly into the computational data analysis, conservatively in a way that honors the complete information of the experimental data, whether or not consistent with the prior expectation. Consistent with analogous results in other fields, we find that the use of available prior knowledge can have a dramatic effect on the resulting molecular weight, sedimentation coefficient, and size-and-shape distributions and can significantly increase both their sensitivity and their resolution. Further, the use of multiple alternative prior information allows us to probe the range of possible interpretations consistent with the data.