Response to Comment on “Laser Desorption/Ionization Coupled to FTICR Mass Spectrometry for Studies of Natural Organic Matter”

Response to Comment on “Laser Desorption/Ionization Coupled to FTICR Mass Spectrometry for Studies of Natural Organic Matter

SHARPER reaction monitoring: generation of a narrow linewidth NMR singlet, without X-pulses, in an inhomogeneous magnetic field

We report a new pure-shift method, termed SHARPER (Sensitive, Homogeneous, And Resolved PEaks in Real time) designed for the analysis of reactions and equilibria by NMR. By focusing on a single selected signal, SHARPER removes all heteronuclear couplings of a selected nucleus without the need to pulse on X channels, thus overcoming hardware limitations of conventional spectrometers. A more versatile decoupling scheme, termed <i>sel</i>-SHARPER, removes all heteronuclear and homonuclear couplings of the selected signal. Both methods are characterized by a periodic inversion of the active spin during the real-time acquisition. In addition to decoupling, they also compensate for pulse imperfections and magnetic field inhomogeneity, generating an extremely narrow singlet with a linewidth approaching limits dictated by the spin–spin relaxation. The decoupling and line narrowing effected by (<i>sel</i>)-SHARPER provide significant increases in the signal-to-noise (S/N) ratio. Increases of 20-fold were routinely achieved for ¹⁹F detection. <i>sel</i>-SHARPER is also applicable to first- and higher-order ¹H spectra. The sensitivity gains are substantially greater for inhomogeneous magnetic fields, including dynamic inhomogeneity caused by gas sparging. The parameters of the pulse sequences have been analyzed in detail to provide guidelines for their most effective application. The considerable reduction in the detection threshold induced by (<i>sel</i>)-SHARPER make the technique particularly suited for <i>in situ</i> monitoring of reaction kinetics. The approach is illustrated by a ¹⁹F NMR study of the protodeboronation of an aryl boronic acid. Here, the high S/N allowed reliable determination of the net protodeoboronation kinetics, and the excess line broadening of ¹⁹F singlets was utilized to characterize the boronic acid/boronate equilibrium kinetics. Oxidation of diphenylphosphine, monitored by ³¹P NMR under optimized gas-flow conditions, demonstrated the high tolerance of SHARPER to dynamic inhomogeneity. The principles of the (<i>sel</i>)-SHARPER sequences are expected to find numerous applications in the design of new NMR experiments

Laser desorption/ionization coupled to FT-ICR mass spectrometry for studies of natural organic matter

Laser desorption/ionization (LDI) was investigated as an ionization method for Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) studies of natural organic matter (NOM). Using International Humic Substances Society standards, Suwannee River fulvic acid (SRFA) and Suwannee River natural organic matter (SRNOM), LDI was found to ionize a very similar set of compounds (>90% of molecular formulas identity) to the matrix assisted laser desorption/ionization (MALDI), while producing higher quality spectra. A comparison of electrospray ionization (ESI) and LDI spectra showed that different types of compounds are ionized by these methods with only 9.9% of molecular formulas common to both. The compounds ionized by LDI/MALDI belong to low oxygen classes (maximum number of species for O7–O9), while ESI compounds belong to higher oxygen classes (maximum number of species for O14–O16). Compounds ionized by LDI can be classified as aliphatic, aromatic, and condensed aromatics in approximately equal measure, while aliphatic compounds dominated the ESI spectra of SRFA. In order to maximize the coverage of molecular species, LDI, as a particularly convenient and readily deployable ionization method, should be used routinely in combination with other ionization methods, such as ESI, for FTICR MS studies of NOM

New F-19 NMR methodology reveals structures of molecules in complex mixtures of fluorinated compounds

Although the number of natural fluorinated compounds is very small, fluorinated pharmaceuticals and agrochemicals are numerous. (19)F NMR spectroscopy has a great potential for the structure elucidation of fluorinated organic molecules, starting with their production by chemical or chemoenzymatic reactions, through monitoring their structural integrity, to their biotic and abiotic transformation and ultimate degradation in the environment. Additionally, choosing to incorporate (19)F into any organic molecule opens a convenient route to study reaction mechanisms and kinetics. Addressing limitations of the existing (19)F NMR techniques, we have developed methodology that uses (19)F as a powerful spectroscopic spy to study mixtures of fluorinated molecules. The proposed (19)F-centred NMR analysis utilises the substantial resolution and sensitivity of (19)F to obtain a large number of NMR parameters, which enable structure determination of fluorinated compounds without the need for their separation or the use of standards. Here we illustrate the (19)F-centred structure determination process and demonstrate its power by successfully elucidating the structures of chloramination disinfectant by-products of a single mono-fluorinated phenolic compound, which would have been impossible otherwise. This novel NMR approach for the structure elucidation of molecules in complex mixtures represents a major contribution towards the analysis of chemical and biological processes involving fluorinated compounds

Comparison of HPLC and NMR for quantification of the main volatile fatty acids in rumen digesta

Accurate quantification of volatile fatty acid (VFA) concentrations in rumen fluid are essential for research on rumen metabolism. The study comprehensively investigated the pros and cons of High-performance liquid chromatography (HPLC) and (1)H Nuclear magnetic resonance ((1)H-NMR) analysis methods for rumen VFAs quantification. We also investigated the performance of several commonly used data pre-treatments for the two sets of data using correlation analysis, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The molar proportion and reliability analysis demonstrated that the two approaches produce highly consistent VFA concentrations. In the pre-processing of NMR spectra, line broadening and shim correction may reduce estimated concentrations of metabolites. We observed differences in results using multiplet of different protons from one compound and identified “handle signals” that provided the most consistent concentrations. Different data pre-treatment strategies tested with both HPLC and NMR significantly affected the results of downstream data analysis. “Normalized by sum” pre-treatment can eliminate a large number of positive correlations between NMR-based VFA. A “Combine” strategy should be the first choice when calculating the correlation between metabolites or between samples. The PCA and PLS-DA suggest that except for “Normalize by sum”, pre-treatments should be used with caution

Solution structure and in Silico binding of a cyclic peptide with hepatitis B surface antigen

A specific ligand targeting the immunodominant region of hepatitis B virus is desired in neutralizing the infectivity of the virus. In a previous study, a disulfide constrained cyclic peptide cyclo S1,S9 Cys-Glu-Thr-Gly-Ala-Lys-Pro-His-Cys (S1, S9-cyclo-CETGAKPHC) was isolated from a phage displayed cyclic peptide library using an affinity selection method against hepatitis B surface antigen. The cyclic peptide binds tightly to hepatitis B surface antigen with a relative dissociation constant (KDrel) of 2.9 nm. The binding site of the peptide was located at the immunodominant region on hepatitis B surface antigen. Consequently, this study was aimed to elucidate the structure of the cyclic peptide and its interaction with hepatitis B surface antigen in silico. The solution structure of this cyclic peptide was solved using 1H, 13C, and 15N NMR spectroscopy and molecular dynamics simulations with NMR-derived distance and torsion angle restraints. The cyclic peptide adopted two distinct conformations due to the isomerization of the Pro residue with one structured region in the ETGA sequence. Docking studies of the peptide ensemble with a model structure of hepatitis B surface antigen revealed that the cyclic peptide can potentially be developed as a therapeutic drug that inhibits the virus–host interactions