Residue-specific NH exchange rates studied by NMR diffusion experiments

We present a novel approach to the investigation of rapid (>2s(-1)) NH exchange rates in proteins, based on residue-specific diffusion measurements. (1)H, (15)N-DOSY-HSQC spectra are recorded in order to observe resolved amide proton signals for most residues of the protein. Human ubiquitin was used to demonstrate the proposed method. Exchange rates are derived directly from the decay data of the diffusion experiment by applying a model deduced from the assumption of a two-site exchange with water and the "pure" diffusion coefficients of water and protein. The "pure" diffusion coefficient of the protein is determined in an experiment with selective excitation of the amide protons in order to suppress the influence of magnetization transfer from water to amide protons on the decay data. For rapidly exchanging residues a comparison of our results with the exchange rates obtained in a MEXICO experiment showed good agreement. Molecular dynamics (MD) and quantum mechanical calculations were performed to find molecular parameters correlating with the exchangeability of the NH protons. The RMS fluctuations of the amide protons, obtained from the MD simulations, together with the NH coupling constants provide a bilinear model which shows a good correlation with the experimental NH exchange rates.     

Optimal control based NCO and NCA experiments for spectral assignment in biological solid-state NMR spectroscopy

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of (13)C,(15)N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically (15)N to (13)C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly (13)C,(15)N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical (15)N-(13)C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D (15)N-->(13)C coherence transfer experiments, as well as 2D and 3D (15)N,(13)C and (15)N,(13)C,(13)C chemical shift correlation experiments on uniformly (13)C,(15)N-labeled ubiquitin.     

LC–MS Quantification of a Sulfosuccinate Surfactant in Agrochemical Formulations

Agrochemical products are a mixture of active ingredient(s) and inerts, which serve as dispersing or wetting agent or as emulsifiers. Varying qualities of these raw materials can have a significant impact on the properties of the final agrochemical product and so its quality has to be controlled. In this work the commercially available mixture of sodium bis(2-ethylhexyl) sulfosuccinate (trade name: Aerosol OT or AOT) and its surface active isomeric by-products sodium 1-carboxy-3-[(2-ethylhexyl)oxy]-3-oxopropane-1-sulfonate and sodium 3-carboxy-1-[(2-ethylhexyl)oxy]-1-oxopropane-2-sulfonate were analyzed. A method using liquid chromatography coupled with time-of-flight mass spectrometry (LC-ToF–MS) with exact mass measurement was developed to quantify these molecules simultaneously. Both by-products were not commercially available and thus were synthesised as analytical standards for method validation. For this purpose, two regio-selective syntheses were developed. Validation was done according to DIN 32645 and recovery and precision for two different matrices were determined. Significant differences were observed in the by-product spectrum of real samples of Aerosol OT of three different suppliers. Their influence on the properties of an agrochemical can now be investigated, as a precise and accurate determination of the target analytes has been developed in this work.     

LC–MS Quantification of a Sulfosuccinate Surfactant in Agrochemical Formulations

Agrochemical products are a mixture of active ingredient(s) and inerts, which serve as dispersing or wetting agent or as emulsifiers. Varying qualities of these raw materials can have a significant impact on the properties of the final agrochemical product and so its quality has to be controlled. In this work the commercially available mixture of sodium bis(2-ethylhexyl) sulfosuccinate (trade name: Aerosol OT or AOT) and its surface active isomeric by-products sodium 1-carboxy-3-[(2-ethylhexyl)oxy]-3-oxopropane-1-sulfonate and sodium 3-carboxy-1-[(2-ethylhexyl)oxy]-1-oxopropane-2-sulfonate were analyzed. A method using liquid chromatography coupled with time-of-flight mass spectrometry (LC-ToF–MS) with exact mass measurement was developed to quantify these molecules simultaneously. Both by-products were not commercially available and thus were synthesised as analytical standards for method validation. For this purpose, two regio-selective syntheses were developed. Validation was done according to DIN 32645 and recovery and precision for two different matrices were determined. Significant differences were observed in the by-product spectrum of real samples of Aerosol OT of three different suppliers. Their influence on the properties of an agrochemical can now be investigated, as a precise and accurate determination of the target analytes has been developed in this work.