Biophysical Technologies

Native nanoESI-MS, FAXS Fluorine chemical shift Anisotropy and eXchange for Screening and DOSY Diffusion Ordered Spectroscopy.

Native nanoESI-MS

This specific application of MS is called supramolecular-, noncovalent-, nondenaturing- or native-MS. This approach is used for the detailed structural and functional characterisation of a wide variety of assemblies consisting of several proteins, proteins and small molecules, nucleic acids, proteins and nucleic acids, as well as nucleic acids and small molecules.

The main strengths of native MS include:

• The sensitivity: detection of picomole amounts of complexes
• The selectivity: species are clearly identified through accurate mass measurements
• The rapidity: analyses take only few minutes
• MS analysis of non covalent complexes does not require any protein and/or ligand labeling
• The ability to produce evidence, on the same mass spectrum, of several assemblies present together in solution

As a consequence, two major applications for native MS have emerged rapidly:

1. Native MS offers a support to x-ray crystallography or NMR studies: with only hundreds of micrograms, it provides an assessment of the purity, homogeneity and integrity of the protein samples and the determination of complex stoichiometry.
2. In early drug research, MS analysis of noncovalent complexes, especially protein/small molecule complexes, is a reliable approach to identify, validate and/or characterise hit and lead molecules.

• Evidence of ligand binding and determination of protein–ligand complex stoichiometry
• Assessment of ligand-binding site specificity
• Determination of ligand-binding affinities and absolute dissociation constants (KD) calculation
• Detection of fortuitous ligand
• Effect of ligand binding on the stability of macromolecular assemblies
• Collision-induced dissociation experiments to assess the type and strength of interaction involved in protein–ligand complexes

Ref: Vivat el al., Future med Chem.,1(2), Jan 2010, pp.35-50(16). Renaud and Delsuc, Curr. Op. Pharmacol., 9(5), Oct 2009, pp 622-628(9).

FAXS Fluorine chemical shift – Anisotropy and eXchange for Screening

The ligand- and substrate-based fluorine NMR approaches are powerful tools for rapidly performing screening against a biomolecular target and determining the KD and IC50 values of the identified hits.

The use of 19F chemical-shift mapping studies to study ligand binding has several advantages:

• Compounds that bind to a given protein can be found and characterised without the need to develop a specific assay or having knowledge of the function of the protein
• This approach can also rapidly provide information on the site of binding
• The chemical shift approach is a very robust, reliable and reproducible ligand binding assays

As well as the ability to detect weak intermolecular interactions makes NMR ideal for fragment-based screening.

Ref: Dalvit, Ligand – and substrate-based 19F NMR screening: Principles and applications to drug discovery; Progress Nuc. Reson. Magn. (2007) pp 243-271. Bartoli et al., The fragment-approach: An update; Drug Discovery: Technologies (2006) pp425-431.

DOSY Diffusion Ordered Spectroscopy

NMR diffusion experiments provide a way to separate the different compounds in a mixture based on the differing translation diffusion coefficients (and therefore differences in the size and shape of the molecule, as well as physical properties of the surrounding environment such as viscosity, temperature, etc) of each chemical species in solution.

In a certain way, it can be regarded as a special chromatographic method for physical component separation, but unlike those techniques, it does not require any particular sample preparation or chromatographic method optimization and maintains the innate chemical environment of the sample during analysis.

NovAliX studies are enabled and powered by a powerful proprietary software for NMR data processing and management for rapid and efficient analysis.

Ref: Assemat et al. Validation of molecular mass measurements by means of Diffusion-Ordered NMR Spectroscopy; Application to Oligosaccharides. CRChimie (2009) pp. 1-8. Balayssac et al. 2D and 3D DOSY 1H NMR, a useful tool for analysis of complex mixtures: Application to herbal drugs or dietary supplements for erectile dysfunction. Journal of Pharmaceutical and Biomedical Analysis (2009) vol. 50 pp. 602-612. Arold et al. Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein. Prot Science (2000) vol. 9 (6) pp. 1137-48. Delsuc and Malliavin. Maximum entropy processing of DOSY NMR spectra. Anal Chem (1998).