scholarly journals US-SOMO HPLC-SAXS module: dealing with capillary fouling and extraction of pure component patterns from poorly resolved SEC-SAXS data

2016 ◽  
Vol 49 (5) ◽  
pp. 1827-1841 ◽  
Author(s):  
Emre Brookes ◽  
Patrice Vachette ◽  
Mattia Rocco ◽  
Javier Pérez
Keyword(s):  
High Performance ◽  
Pure Component ◽  
Size Exclusion ◽  
Baseline Correction ◽  
Saxs Data ◽  
X Ray ◽  
Exclusion Chromatography ◽  
Flow Through ◽  
Gaussian Decomposition ◽  
Comprehensive Framework

Size-exclusion chromatography coupled with SAXS (small-angle X-ray scattering), often performed using a flow-through capillary, should allow direct collection of monodisperse sample data. However, capillary fouling issues and non-baseline-resolved peaks can hamper its efficacy. The UltraScan solution modeler (US-SOMO) HPLC-SAXS (high-performance liquid chromatography coupled with SAXS) module provides a comprehensive framework to analyze such data, starting with a simple linear baseline correction and symmetrical Gaussian decomposition tools [Brookes, Pérez, Cardinali, Profumo, Vachette & Rocco (2013). J. Appl. Cryst. 46, 1823–1833]. In addition to several new features, substantial improvements to both routines have now been implemented, comprising the evaluation of outcomes by advanced statistical tools. The novel integral baseline-correction procedure is based on the more sound assumption that the effect of capillary fouling on scattering increases monotonically with the intensity scattered by the material within the X-ray beam. Overlapping peaks, often skewed because of sample interaction with the column matrix, can now be accurately decomposed using non-symmetrical modified Gaussian functions. As an example, the case of a polydisperse solution of aldolase is analyzed: from heavily convoluted peaks, individual SAXS profiles of tetramers, octamers and dodecamers are extracted and reliably modeled.

scholarly journals Fibrinogen species as resolved by HPLC-SAXS data processing within theUltraScan Solution Modeler(US-SOMO) enhanced SAS module

2013 ◽  
Vol 46 (6) ◽  
pp. 1823-1833 ◽  
Author(s):  
Emre Brookes ◽  
Javier Pérez ◽  
Barbara Cardinali ◽  
Aldo Profumo ◽  
Patrice Vachette ◽  
...  
Keyword(s):  
User Interfaces ◽  
High Performance ◽  
Weight Fraction ◽  
Size Exclusion ◽  
High Molecular Weight Fraction ◽  
Saxs Data ◽  
Cross Sectional ◽  
Baseline Drift ◽  
Gaussian Decomposition ◽  
Automated Procedures

Fibrinogen is a large heterogeneous aggregation/degradation-prone protein playing a central role in blood coagulation and associated pathologies, whose structure is not completely resolved. When a high-molecular-weight fraction was analyzed by size-exclusion high-performance liquid chromatography/small-angle X-ray scattering (HPLC-SAXS), several composite peaks were apparent and because of the stickiness of fibrinogen the analysis was complicated by severe capillary fouling. Novel SAS analysis tools developed as a part of theUltraScan Solution Modeler(US-SOMO; http://somo.uthscsa.edu/), an open-source suite of utilities with advanced graphical user interfaces whose initial goal was the hydrodynamic modeling of biomacromolecules, were implemented and applied to this problem. They include the correction of baseline drift due to the accumulation of material on the SAXS capillary walls, and the Gaussian decomposition of non-baseline-resolved HPLC-SAXS elution peaks. It was thus possible to resolve at least two species co-eluting under the fibrinogen main monomer peak, probably resulting from in-column degradation, and two others under an oligomers peak. The overall and cross-sectional radii of gyration, molecular mass and mass/length ratio of all species were determined using the manual or semi-automated procedures available within theUS-SOMOSAS module. Differences between monomeric species and linear and sideways oligomers were thus identified and rationalized. This newUS-SOMOversion additionally contains several computational and graphical tools, implementing functionalities such as the mapping of residues contributing to particular regions ofP(r), and an advanced module for the comparison of primaryI(q)versus qdata with model curves computed from atomic level structures or bead models. It should be of great help in multi-resolution studies involving hydrodynamics, solution scattering and crystallographic/NMR data.

Distinct Instrumental Approaches to SAXS

2018 ◽  
Author(s):  
Eaton E. Lattman ◽  
Thomas D. Grant ◽  
Edward H. Snell
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Size Exclusion ◽  
Saxs Data ◽  
Sample Handling ◽  
X Ray ◽  
Macromolecular Conformation ◽  
Exclusion Chromatography ◽  
Membrane Bound ◽  
Instrumental Approaches

There are more specialized applications of SAXS and SANS which require specific experimental considerations. This chapter covers size exclusion chromatography which has proven to be useful to study both soluble and membrane bound proteins allowing the study of samples that show time and concentration dependent dynamics. It also describes iime-resolved techniques for SAXS and in a few cases, SANS. Finally, with improved X-ray sources, detectors, sample handling, and compute power, the ability to perform SAXS data in high-throughput is available. This is discussed in enabling the use of SAXS to study protein interactions, map macromolecular conformation, and rapidly characterize samples amongst other applications.

scholarly journals Methods for analysis of size-exclusion chromatography–small-angle X-ray scattering and reconstruction of protein scattering

2015 ◽  
Vol 48 (4) ◽  
pp. 1102-1113 ◽  
Author(s):  
Andrew W. Malaby ◽  
Srinivas Chakravarthy ◽  
Thomas C. Irving ◽  
Sagar V. Kathuria ◽  
Osman Bilsel ◽  
...  
Keyword(s):  
Linear Combination ◽  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Data Sets ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Ray Scattering

Size-exclusion chromatography in line with small-angle X-ray scattering (SEC–SAXS) has emerged as an important method for investigation of heterogeneous and self-associating systems, but presents specific challenges for data processing including buffer subtraction and analysis of overlapping peaks. This paper presents novel methods based on singular value decomposition (SVD) and Guinier-optimized linear combination (LC) to facilitate analysis of SEC–SAXS data sets and high-quality reconstruction of protein scattering directly from peak regions. It is shown that Guinier-optimized buffer subtraction can reduce common subtraction artifacts and that Guinier-optimized linear combination of significant SVD basis components improves signal-to-noise and allows reconstruction of protein scattering, even in the absence of matching buffer regions. In test cases with conventional SAXS data sets for cytochrome c and SEC–SAXS data sets for the small GTPase Arf6 and the Arf GTPase exchange factors Grp1 and cytohesin-1, SVD–LC consistently provided higher quality reconstruction of protein scattering than either direct or Guinier-optimized buffer subtraction. These methods have been implemented in the context of a Python-extensible Mac OS X application known asData Evaluation and Likelihood Analysis(DELA), which provides convenient tools for data-set selection, beam intensity normalization, SVD, and other relevant processing and analytical procedures, as well as automated Python scripts for common SAXS analyses and Guinier-optimized reconstruction of protein scattering.

scholarly journals Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

Crystals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 975
Author(s):  
Melissa A. Graewert ◽  
Stefano Da Vela ◽  
Tobias W. Gräwert ◽  
Dmitry S. Molodenskiy ◽  
Clément E. Blanchet ◽  
...  
Keyword(s):  
Light Scattering ◽  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Set Up ◽  
Ray Scattering

We describe the updated size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) set-up used at the P12 bioSAXS beam line of the European Molecular Biology Laboratory (EMBL) at the PETRAIII synchrotron, DESY Hamburg (Germany). The addition of size exclusion chromatography (SEC) directly on-line to the SAXS capillary has become a well-established approach to reduce the effects of the sample heterogeneity on the SAXS measurements. The additional use of multi-angle laser light scattering (MALLS), UV absorption spectroscopy, refractive index (RI), and quasi-elastic light scattering (QELS) in parallel to the SAXS measurements enables independent molecular weight validation and hydrodynamic radius estimates. This allows one to address sample monodispersity as well as conformational heterogeneity. The benefits of the current SEC-SAXS set-up are demonstrated on a set of selected standard proteins. The processed SEC-SAXS data and models are provided in the Small Angle Scattering Biological Data Bank (SASBDB) and are labeled as “bench-marked” datasets that include the unsubtracted data frames spanning the respective SEC elution profiles and corresponding MALLS-UV-RI-QELS data. These entries provide method developers with datasets suitable for testing purposes, in addition to an educational resource for SAS data analysis and modeling.

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

Sign in / Sign up

Export Citation Format

Share Document